Zhang2020 Book ProteinPhosphorylationAndMeatQ

Dequan Zhang · Xin Li · Li Chen
Chengli Hou · Zhenyu Wang
Protein
Phosphorylation
and Meat
Quality
Protein Phosphorylation and Meat Quality
Dequan Zhang • Xin Li • Li Chen •
Chengli Hou • Zhenyu Wang
Protein Phosphorylation
and Meat Quality
Dequan Zhang Xin Li
Institute of Food Science and Institute of Food Science and
Technology, Chinese Academy Technology, Chinese Academy
of Agricultural Sciences of Agricultural Sciences
Beijing, China Beijing, China
Li Chen Chengli Hou

Institute of Food Science and Institute of Food Science and
Technology, Chinese Academy Technology, Chinese Academy
of Agricultural Sciences of Agricultural Sciences
Beijing, China Beijing, China
Zhenyu Wang
Institute of Food Science and
Technology, Chinese Academy
of Agricultural Sciences
Beijing, China
ISBN 978-981-15-9440-3 ISBN 978-981-15-9441-0 (eBook)

https://doi.org/10.1007/978-981-15-9441-0
© Springer Nature Singapore Pte Ltd. 2020

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Preface
Meat quality is an important characteristic that determines the value of meat products
and economic benefits. Although research has been performed on different aspects
of meat quality, meat quality issues still exist because meat quality is complex and
controlled by many factors and mechanisms. Protein phosphorylation is regarded as
one of the most common protein post-translational modifications that is considered
universal across all the domains of life. In the field of meat science, the role of
protein phosphorylation in meat quality characteristics is gaining great interest and
more studies are being conducted. This book is based on the achievements of our
team research focusing on protein phosphorylation and meat quality. Our aim was to
provide new ideas for exploring the mechanism of meat quality and possible
solutions for improving meat quality.
The book is divided into three parts preceded by an introduction and background
chapter providing an overview of meat quality mechanism, protein post-translational
modification on meat quality, and current research of protein phosphorylation in live
and postmortem. The first section (Chaps. 2–5) gave a brief introduction of the
methods used for protein phosphorylation detection and discussed the relationship
between protein phosphorylation and meat quality attributes of color, tenderness,
and water holding capacity. The second section (Chaps. 6–9) elucidated possible
mechanisms of the effect of protein phosphorylation on meat quality from the
aspects of glycolysis, myofibril protein degradation, calpain activity, and myoglobin.
The third section (Chaps. 10–12) focused on the improvement of meat quality by
regulating protein phosphorylation, which included the effects of temperature and
ionic strength on protein phosphorylation and three novel technologies.
We wish to express our gratitude to all the contributors to this book, including
Meng Li, Zheng Li, Lijuan Chen and Fan He for their contributions on the studies
related with Part I, Ying Wang, Zheng Li, Manting Du and Meng Li for their
contributions on the studies related with Part II, Chi Ren, Caixia Zhang, Chunhui
Zhang, Yan Zhang and Weiwei Xu for their contributions on the studies related with
v
vi Preface
Part III, Manting Du, Ying Wang, Chi Ren, Lichuang Cao, Muawuz Ijaz, Yingxin
Zhao, Caiyan Huang, Yejun Zhang, Xiangru Wei, Xu Wang, Yujun Xu, Yue Ge,
Dan Jiao and Tongjing Yan for their contributions on preparation of this book,
Caiwei Liu for her contributions on proof of the language. We also want to thank the
production team at Springer for their kind assistance in this book.
Beijing, China Dequan Zhang

Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Meat Quality and Mechanism from Muscle to Meat . . . . . . . . . 1
1.1.1 Importance of Meat Quality . . . . . . . . . . . . . . . . . . . . 1
1.1.2 Mechanism of Meat Quality Formation During
Postmortem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Protein Post-Translational Modification on Meat Quality . . . . . . 3
1.2.1 Definition and the Role of Protein Post-Translational
Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.2 Role of Protein Post-Translational Modification
on Meat Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Current Research of Protein Phosphorylation
(Live and Postmortem) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Part I Relationship Between Protein Phosphorylation and Meat Quality

2 Protein Phosphorylation Detection Method . . . . . . . . . . . . . . . . . . . 11
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Principle of Phosphorylated Protein Detection . . . . . . . . . . . . . . 12
2.3 Gel-Based Phosphorylated Protein Detecting Methods . . . . . . . . 14
2.3.1 Separation of Proteins by 2DE Gel . . . . . . . . . . . . . . . 14
2.3.2 Detection of Phosphorylated Protein by Pro-Q
Diamond Gel Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3.3 Detection of Phosphorylated Protein by Phos-Tag
Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4 Detection of Phosphorylated Proteins by LC-MS/MS . . . . . . . . 17
2.4.1 Labeling Treatment of Samples . . . . . . . . . . . . . . . . . . 17
2.4.2 Label Free Treatment of Samples . . . . . . . . . . . . . . . . 18
2.4.3 Enrichment of Phosphorylated Peptides Using the
TiO2 Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4.4 LC-MS/MS Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 19
vii
viii Contents
2.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3 Protein Phosphorylation Affects Meat Color . . . . . . . . . . . . . . . . . . 23
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2 Changes of Meat Color After Protein Phosphorylation
Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.2.1 Global Level of Protein Phosphorylation After
Regulation of Protein Phosphorylation . . . . . . . . . . . . . 25
3.2.2 Color of Meat with Different Protein Phosphorylation
Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.3 Role of pH and Lactic Acid Content on Color
of Meat with Different Protein Phosphorylation Level . . 28
3.3 Pattern of Sarcoplasmic Protein Phosphorylation in Meat
with Different Color Stability . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3.1 Global Sarcoplasmic Protein Phosphorylation Level
of Meat with Different Color Stability . . . . . . . . . . . . . 31
3.3.2 Correlation of Meat Color Parameters and Sarcoplasmic
Protein Phosphorylation Level . . . . . . . . . . . . . . . . . . . 32
3.3.3 Identification of Phosphorylated Sarcoplasmic
Proteins Related with Meat Color . . . . . . . . . . . . . . . . 37
3.4 Quantitative Phosphoproteome of Meat with Different Color . . . 40
3.4.1 Differentially Expressed Phosphoproteins Between
Meat with Different Color . . . . . . . . . . . . . . . . . . . . . . 40
3.4.2 Functional Enrichment Analysis of Differentially
Expressed Phosphoproteins . . . . . . . . . . . . . . . . . . . . . 42
3.4.3 Functional Annotation Analysis of Key Color-Related
Phosphoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4 Protein Phosphorylation Affects Meat Tenderness . . . . . . . . . . . . . 49
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.2 Changes of Proteins Phosphorylation Levels in Postmortem
Muscle with Different Tenderness . . . . . . . . . . . . . . . . . . . . . . 51
4.2.1 Changes of Sarcoplasmic Proteins Phosphorylation
Levels in Postmortem Muscle with Different
Tenderness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2.2 Changes of Myofibrillar Proteins Phosphorylation
Levels in Postmortem Muscle with Different
Tenderness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.2.3 Functional Verification of Protein Phosphorylation
Regulation of Myofibrillar Protein . . . . . . . . . . . . . . . . 67
4.3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Contents ix
5 Protein Phosphorylation Affects Meat Water Holding Capacity . . . 77

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
5.2 Meat Quality Traits with Different Drip Loss Values . . . . . . . . . 78
5.2.1 Comparison of Physical and Chemical Indicators
Between Different Drip Loss Meat . . . . . . . . . . . . . . . 78
5.2.2 Comparison of Muscle Fiber Ultrastructure Between
Different Drip Loss Meat . . . . . . . . . . . . . . . . . . . . . . 78
5.3 Changes of Proteins Phosphorylation Levels Meat
with Different Drip Loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
5.3.1 Comparison of Sarcoplasmic Proteins Phosphorylation
Levels Between Different Drip Loss Meat . . . . . . . . . . 82
5.3.2 Comparison of Myofibrillar Proteins Phosphorylation
Levels Between Different Drip Loss Meat . . . . . . . . . . 85
5.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Part II Mechanism of the Effect of Protein Phosphorylation

on Meat Quality
6 Mechanism of the Effect of Protein Phosphorylation
on Postmortem Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6.2 The Effect of Sarcoplasmic Protein Phosphorylation
on Glycolysis in Postmortem Muscle . . . . . . . . . . . . . . . . . . . . 95
6.2.1 Global Phosphorylation Level of Sarcoplasmic
Protein with Different Glycolytic Rates . . . . . . . . . . . . 95
6.2.2 Phosphorylation Analysis of Individual Protein
Bands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
6.2.3 Sarcoplasmic Protein Identification with Different
Phosphorylation Level of the Three Glycolytic Rate
Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
6.3 Quantitative Phosphoproteomic Analysis of Muscle
with Different Postmortem Glycolytic Rate . . . . . . . . . . . . . . . . 102
6.3.1 Phosphoprotein Identification and Motif Analysis . . . . . 102
6.3.2 Hierarchical Clustering Analysis . . . . . . . . . . . . . . . . . 104
6.3.3 Functional Enrichment Analysis . . . . . . . . . . . . . . . . . 105
6.3.4 Quantitative Analysis of Phosphopeptides . . . . . . . . . . 105
6.4 Validation of Protein Phosphorylation on Glycolysis
and the Regulation Mechanism of Enzyme Activity . . . . . . . . . . 109
6.4.1 Global Phosphorylation of Sarcoplasmic Protein
with the Regulation of Kinase or Phosphatase
Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.4.2 Glycolytic Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
6.4.3 Gel Band Identification . . . . . . . . . . . . . . . . . . . . . . . . 111
x Contents
6.4.4 Glycolytic Enzymes Activities and Phosphorylation

Level After Regulation . . . . . . . . . . . . . . . . . . . . . . . . 113
6.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
7 Mechanism of the Effect of Protein Phosphorylation on Myofibril
Protein Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.2 The Effect of Protein Phosphorylation on Myosin and Actin
Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7.2.1 Global Phosphorylation Level of Myofibrillar Protein . . 125
7.2.2 Degradation of Myosin and Actin . . . . . . . . . . . . . . . . 126
7.3 Phosphorylation Prevents Myosin and Actin Degradation
by μ-Calpain In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
7.3.1 Phosphorylation Level of Myosin Heavy Chain
and Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
7.3.2 Myosin and Actin Degradation by μ-Calpain
at Different Ca2+ Concentration . . . . . . . . . . . . . . . . . . 131
7.4 Influence Mechanism of Protein Phosphorylation on Titin
Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
7.4.1 Changes in pH Value of the Three Ovine Muscles . . . . 134
7.4.2 Changes in Phosphorylation Level of Titin . . . . . . . . . . 135
7.4.3 Degradation of Titin . . . . . . . . . . . . . . . . . . . . . . . . . . 135
7.5 Effects of Phosphorylation on Titin Degradation at Different
Ca2+ Concentrations Incubation In Vitro . . . . . . . . . . . . . . . . . . 137
7.5.1 Phosphorylation and Dephosphorylation of Titin . . . . . 138
7.5.2 pH Values of Incubation Systems . . . . . . . . . . . . . . . . 139
7.5.3 Degradation of Titin . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7.6 Influence of Protein Phosphorylation on Other Myofibril
Proteins Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7.6.1 Troponin T (TnT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7.6.2 Desmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
7.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
8 Mechanism of the Effect of Protein Phosphorylation on Calpain
Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
8.2 Relationship Between Protein Phosphorylation and Calpain
Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
8.2.1 Changes in pH in Mutton with Different Tenderness
at Postmortem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
8.2.2 Phosphorylation Level of Sarcoplasmic Proteins
During Postmortem . . . . . . . . . . . . . . . . . . . . . . . . . . 151
8.2.3 Casein Zymography Analysis of μ-/m-Calpain . . . . . . . 152
Contents xi
8.2.4 The Degradation of μ-Calpain 80 kDa Subunit . . . . . . . 153

8.2.5 The Degradation of Calpastatin . . . . . . . . . . . . . . . . . . 154
8.3 Effects of Phosphorylation of Sarcoplasmic Proteins
on μ-Calpain Activity at Different Incubation Temperature . . . . 155
8.3.1 Phosphorylation Level Analysis of Sarcoplasmic
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
8.3.2 μ- and m-Calpain Activity . . . . . . . . . . . . . . . . . . . . . . 156
8.4 Effects of Phosphorylation on μ-Calpain Activity at Different
Incubation Temperature In Vitro . . . . . . . . . . . . . . . . . . . . . . . 160
8.4.1 The pH Values of μ-Calpain Solution Measured
Before and After Treated with AP/PKA . . . . . . . . . . . . 160
8.4.2 The Phosphorylation Level of μ-Calpain . . . . . . . . . . . 161
8.4.3 The Degradation of μ-Calpain . . . . . . . . . . . . . . . . . . . 164
on μ-Calpain Activity at Different Ca2+ Concentrations . . . . . . . 165
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
8.5.2 μ-Calpain Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
8.6 Effects of Phosphorylation on μ-Calpain Activity at Different
Ca2+ Concentrations In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . 170
8.6.1 The Changes in pH Values During Incubation . . . . . . . 170
8.6.2 The Phosphorylation of μ-Calpain . . . . . . . . . . . . . . . . 170
8.6.3 The Degradation of μ-Calpain . . . . . . . . . . . . . . . . . . . 171
8.6.4 Changes in μ-Calpain Secondary Structure . . . . . . . . . . 174
8.6.5 Detection of Phosphorylated Peptides and
Phosphorylated Sites of μ-Calpain . . . . . . . . . . . . . . . . 175
8.6.6 The Ser-Phosphorylation Level of μ-Calpain . . . . . . . . 177
8.7 The Inhibition of Calpastatin to the Activity
of Phosphorylated μ-Calpain . . . . . . . . . . . . . . . . . . . . . . . . . . 177
8.7.1 The pH Values of μ-Calpain Solution Measured
Before and After Treated with AP/PKA . . . . . . . . . . . . 178
8.7.2 Phosphorylation Level of Heat Stable Proteins
and μ-Calpain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
8.7.3 The Activity of μ-Calpain . . . . . . . . . . . . . . . . . . . . . . 180
8.7.4 The Degradation of Calpastatin . . . . . . . . . . . . . . . . . . 185
8.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
9 Mechanism of the Effect of Protein Phosphorylation on
Myoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
9.2 Changes of Myoglobin Relative Contents in Meat After
Regulation of Protein Phosphorylation . . . . . . . . . . . . . . . . . . . 192
9.3 Changes of Myoglobin Redox Stability After Regulation
of Protein Phosphorylation In Vitro . . . . . . . . . . . . . . . . . . . . . 196
9.3.1 Changes of Myoglobin Phosphorylation Level . . . . . . . 196
xii Contents
9.3.2 Changes of Myoglobin Relative Contents . . . . . . . . . . 198

9.3.3 Changes of pH in Myoglobin Incubation System . . . . . 198
9.3.4 Changes of Myoglobin Secondary Structure . . . . . . . . . 200
9.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Part III Improvement of Meat Quality by Regulating Protein

Phosphorylation
10 Effects of Temperature on Protein Phosphorylation . . . . . . . . . . . . 207
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
10.2 Effects of Temperature on Protein Phosphorylation in
Postmortem Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
10.2.1 Effect of Temperature on Glycolysis in Postmortem
Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
10.2.2 Effect of Temperature on ATP Content in Postmortem
Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
10.2.3 Effect of Temperature on Global Phosphorylation
Levels in Postmortem Muscle . . . . . . . . . . . . . . . . . . . 211
10.2.4 Association between Phosphorylation Levels and
Temperature, pH, ATP Content . . . . . . . . . . . . . . . . . . 214
10.2.5 Identification of Individual Protein Bands . . . . . . . . . . 214
10.3 Effect of ATP on Protein Phosphorylation in Postmortem
Muscle with Different Temperatures . . . . . . . . . . . . . . . . . . . . . 219
Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
10.3.2 Effect of ATP on pH in Postmortem Muscle . . . . . . . . 221
10.3.3 Effect of ATP on Global Phosphorylation Levels in
Postmortem Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . 221
10.3.4 Effect of ATP on Protein Degradation in Postmortem
Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
10.4 Effect of Temperature and pH on Dephosphorylation
of Myofibrillar Protein In Vitro . . . . . . . . . . . . . . . . . . . . . . . . 227
10.4.1 Effect of Temperature and pH on the Activity
of Alkaline Phosphatase . . . . . . . . . . . . . . . . . . . . . . . 227
10.4.2 Effect of Temperature and pH on Phosphorylation
Level of Myofibrillar Protein . . . . . . . . . . . . . . . . . . . . 229
Level of AP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
10.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
11 Effects of Ionic Strength on Protein Phosphorylation . . . . . . . . . . . 237
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
11.2 Phosphorylation Level and Influencing Pathway
of Myofibrillar and Sarcoplasmic Proteins of Muscle
in Response to Salting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Contents xiii
11.2.1 Global Phosphorylation of Myofibrillar Proteins with

Salting Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
11.2.2 Global Phosphorylation of Sarcoplasmic Proteins with
Salting Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
11.2.3 Salting Influences the Phosphorylation of Glycolytic
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
11.2.4 Salting Regulates the Phosphorylation of Heat Shock
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
11.2.5 Research Prospect . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
11.3 Identification of Specific Phosphorylated Proteins Induced by
Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
11.3.1 Identification of Differentially Phosphorylated Proteins
After Salting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
11.3.2 Differential Phosphorylated Proteins Regulating
Glycolysis Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 252
11.3.3 Differential Phosphorylated Proteins Changing the
Protein Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
11.3.4 Differential Phosphorylated Proteins Regulating the
Muscle Contractile and Protein Dissociation . . . . . . . . . 256
11.4 Effect of Salting Temperature on the Protein Phosphorylation
of Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
11.4.1 Effect of Salting Temperature on the Myofibrillar
Protein Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . 257
11.4.2 Effect of Salting Temperature on the Sarcoplasmic
Protein Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . 261
11.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
12 Improvement of Meat Quality by Novel Technology . . . . . . . . . . . . 271
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
12.2 Controlled Freezing Point Storage . . . . . . . . . . . . . . . . . . . . . . 272
12.2.1 Effect of Controlled Freezing Point Storage on Meat
Color Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
12.2.2 Effect of Controlled Freezing Point Storage on Protein
Kinases’ Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
12.2.3 Effect of Controlled Freezing Point Storage on
Phosphorylation Levels of Sarcoplasmic Protein . . . . . . 278
12.2.4 Effect of Controlled Freezing Point Storage on
Phosphorylation Levels of Myofibrillar Protein . . . . . . . 280
12.3 Low-Variable Temperature and High Humidity Thawing . . . . . . 282
12.3.1 Effect of Low-Variable Temperature
and High Humidity Thawing on Meat Quality
Color, Thawing Loss and Cooking Loss, Texture . . . . . 282
12.3.2 Effect of Low-Variable Temperature and High
Humidity Thawing on Myofibril Protein . . . . . . . . . . . 286
xiv Contents

Humidity Thawing on the Microstructure of Meat . . . . 287
12.4 Pulse Pressure Pickling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
12.4.1 Effect of Pulse Pressure Salting on Meat Quality . . . . . 288
12.4.2 Effect of Pulse Pressure Salting on Myofibrillar and
Sarcoplasmic Protein . . . . . . . . . . . . . . . . . . . . . . . . . 295
12.4.3 Effect of Pulse Pressure Salting on Microstructure of
Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
12.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Chapter 1
Introduction
1.1 Meat Quality and Mechanism from Muscle to Meat
1.1.1 Importance of Meat Quality
Meat is a kind of important agricultural product and how to maximize meat quality is
an urgent issue as it is of great concern of every person involved with the meat
industry, including farmers, producers, consumers, and scientists. Interestingly, the
definition of meat quality changes from person to person. For producers, meat
quality is mainly determined by not only the parameters demanded by the consumer
but also the objective factors representing the industrial meat characteristics, includ-
ing meat weight losses. For the supply chain, pH, color, water holding capacity
(WHC), cooking loss, and tenderness all are important for integrated quality and
homogenous products. Consumers mainly focus on color and tenderness and some
are conscious of the fat contents. Above these, for scientists, all these parameters are of
equal importance. All the meat quality parameters are interconnected and dependent
on many intrinsic (innate to the animals, such as breed, genetics, and age) and extrinsic
(related with the pre- and postmortem management and environment) factors.
With the increased great demand of meat, the awareness of consumer from all of
the world has transformed into meat quality and safety (Taheri-Garavand et al.
2019). Generally, meat quality includes five aspects: eating quality, nutritional
quality, technological quality, safety quality, and humane quality (Zhou et al.
2007). Eating quality mainly involves color, tenderness, flavor, juiciness, water
holding capacity, etc. The nutrition quality consists of contents of water, protein,
fat, etc. The different kinds of fatty acid profiles lead to various sensory traits of
meat. The status of shortening, rigor, the content of connective tissue, the degree of
protein denaturation, the ability of oxidation and/or pH value affect the technological
quality of meat. Microbial spoilage can evaluate the freshness of meat by using the
figure of total volatile basic nitrogen (TVB-N) or aerobic bacterial count. The
© Springer Nature Singapore Pte Ltd. 2020 1

D. Zhang et al., Protein Phosphorylation and Meat Quality,
https://doi.org/10.1007/978-981-15-9441-0_1
2 1 Introduction
residual veterinary drugs, pesticides and heavy metals are also factors that have large
negative effects on meat safety quality. With the increasing awareness of protecting
animals and environment, humane quality of meat has become a new aspect (Warriss
2000). As a result, the system and condition of animal feeding are friendlier and of
less stress. However, the total meat quality is affected not only by these five aspects
but also innate traits like breed and age. Dhanda et al. (2003) studied six goat
genotypes and the results showed that the subcutaneous fat, shear force, juiciness,
and other sensory characteristics were different in these goats. Therefore, meat
quality is mainly affected by the animal species, age, sex, physiological state, and
postmortem factors including postmortem muscle biochemistry and conversion of
muscle into meat.
1.1.2 Mechanism of Meat Quality Formation During

Postmortem
Before slaughtering, energy for muscle contraction and relaxation derives from the
adenosine triphosphate (ATP) through mitochondrial oxidative metabolism, phos-
phocreatine, and myokinase (Scheffler and Gerrard 2007). But after animal
slaughtering, the supply of oxygen to the muscles stops and oxidative decarboxyl-
ation and phosphorylation no longer operate. At this point, phosphocreatine is the
main supplier of ATP. After most of the phosphocreatine are depleted, the level of
ATP content decreases quickly. The glycogen in the muscle is degraded and a small
number of ATP are produced through glycolysis. As a result, lactic acid generates
and pH value declines, consequently acidifying the environment, leading to the
conversion of muscles into the meat. This acid declines the pH from neutral muscle
pH (7.0) to the acidic meat pH (5.5). When muscle is stored at high temperature, it is
easy to become pale, soft, exudative (PSE). After electrical stimulation, pH value
declines rapidly and PSE-like traits are generated (Bowker et al. 1999). With ATP
consumption, the actomyosin cross-bridge starts to form, which is the signal of rigor
mortis. When ATP content is extremely consumed, the process of rigor finishes.
With storage time increasing, muscle tenderness becomes tender because of the
degradation of structural myofibrillar proteins.
During the process of muscle to meat, meat tenderness, color, and WHC change a
lot. Tenderness is an important feature of meat quality, having a close relationship
with WHC. When actomyosin cross-bridge cannot be destructed, it reaches the
largest tension of muscle and the least WHC. With the release of enough Ca2+,
calpain proteolytic enzymes improve some structural myofibrillar proteins degrade,
leading to tenderization (Ertbjerg and Puolanne 2017). Meat color is another impor-
tant sensory feature and myoglobin (Mb) is the main protein which controls the meat
color. Many factors such as glycogen storage, chilling rate, antioxidant accumula-
tion, genetics, diet, etc. affect meat color. The physical, chemical, and biological
reactions are complex in postmortem muscle, many quality traits are impressionable,
so keeping the best meat quality is a meaningful subject.
1.2 Protein Post-Translational Modification on Meat Quality 3
1.2 Protein Post-Translational Modification on Meat

Quality
1.2.1 Definition and the Role of Protein Post-Translational

Modification
Post-translational modifications, referring to the covalent enzymatic modifications of

proteins, are mainly responsible for all the biological and biochemical mechanisms
by regulating proteins function via modulating their stability and shape. To under-
stand the biochemistry of any mechanism, analysis of these protein modifications is
essential. The main post-translational modifications are protein phosphorylation,
acetylation, ubiquitination, nitrosylation, succinylation, etc.
The protein phosphorylation is a process, where the transfer of phosphate of ATP
or guanosine triphosphate (GTP) to the hydroxyl groups of its proteins under protein
kinases and reversion by the counterpart phosphatases. The role of phosphatases is
to catalyze the transfer of phosphate of phosphoprotein to the water molecule
(Ubersax and Ferrell 2007). Although both protein kinase and phosphatase are
phosphotransferases, they catalyze opposite processes to regulate the functions of
many cellular proteins ranging from the fate of cell to regulation of metabolism.
Protein phosphorylation is the most common type of post-translational modification
and essentially affects every basic cellular process.
Acetylation, controlled by acetyltransferases enzymes which transfer the acetyl
group from a specific amino acid to either the α-amino group of amino-terminal
residues or the ε-amino group of lysine residues at various positions, changes the
structure, molecular weight, and ultimately the function of targeted molecule
(Polevoda and Sherman 2002). Most of the enzymes involved in anabolism and
catabolism have been shown to be acetylated (Guan and Xiong 2011).
S-nitrosylation is a nitric oxide (NO) signaling pathway independent of guano-
sine 30 , 50 -cyclic phosphate (cGMP), and NO plays an important role as a signaling
molecule and covalently binds to free mercaptocysteine residues of certain proteins
to form S-nitrosothiol (SNO) (Foster et al. 2009). The products of SNO have been
observed to regulate protein conformation, activity, and function (Hess et al. 2005).
The NO and NO-induced S-nitrosylation take part in glucose uptake, muscle con-
traction, and muscular dystrophies in skeletal muscles by managing cGMP and
ryanodine receptor 1 (Stamler and Meissner 2001), and subsets of S-nitrosylated
proteins play a major role in the regulation of tissue homeostasis (Furuta 2017).
Ubiquitination means the addition of ubiquitin to a substrate protein, and
ubiquitin is a small regulatory protein (8.6 kDa) found in most tissues of eukaryotic
organisms (Majetschak 2011). Ubiquitination is crucial for protein localization,
metabolism, function, regulation, and degradation (Schreiweis et al. 2005).
Succinylation is a recently discovered novel post-translational modification in
which succinyl-CoA modifies protein lysine groups (Yang and Gibson 2019). This
modification is found in many proteins, including histones (Xie et al. 2012). The
potential role of succinylation is still under investigation, but as the addition of
4 1 Introduction
succinyl group changes lysine’s charge from +1 to 1 and introduces a relatively

large structural moiety (100 Da), bigger than acetylation (42 Da), it is expected that
succinylation can lead to more significant changes in protein structure and function
than that of acetylation (Zhang et al. 2011).
1.2.2 Role of Protein Post-Translational Modification

on Meat Quality
Meat quality is an important aspect that determines the value and economic benefit
of meat products. To obtain high quality meat, it is necessary to clarify the changes
and mechanisms in the process of meat quality formation. To better understand the
mechanism of meat quality regulation, proteomics has been widely used to describe
the mechanism of meat quality formation and to explore its biomarkers (Lametsch
and Bendixen 2001; Lametsch et al. 2003; Mekchay et al. 2010). The mentioned
protein modifications above have a significant effect on the development of ultimate
meat quality. Recently, it was found out that protein phosphorylation showed a
significant effect on the color, tenderness, and WHC of meat. Li et al. (Li et al.
2017a, b, c) measured the phosphorylation level of myofibrillar proteins in different
tenderness groups and examined the phosphoproteomes in ovine muscle with
different degrees of tenderness over time. Most of the different phosphoproteins
maintained sarcomeric functions or were involved in glycometabolism. Allison et al.
(2003) believed that protein phosphorylation could improve muscle glycolysis and
reduce postmortem proteolysis, which had adverse effects on meat color, WHC, and
tenderness. Li et al. (2017a, b, c) obtained the sarcoplasmic proteins with different
phosphorylation levels by adding phosphates and protein kinase inhibitors to ground
lamb meat and investigated their effects on meat color stability. They discovered that
the global phosphorylation level of sarcoplasmic protein was inversely related to
lamb color stability and proposed that protein phosphorylation can influence meat
color by regulating the glycolytic enzymes and redox form of myoglobin. Scheffler
and Gerrard (2007) found out that the phosphorylation levels of rate-limiting
enzymes, such as glycogen phosphorylase (GP), phosphofructokinase (PFK), pyru-
vate kinase (PK), etc., mainly controlled the glycolysis, protein degradation, and
ultimately the meat fluid losses. Likewise, protein acetylation regulated the post-
mortem muscle glycolysis (Li et al. 2017a, b, c) and it suggested that 50 adenosine
monophosphate-activated protein kinase (AMPK) can be targeted to control the
postmortem metabolism. Therefore, by doing so, pale, soft, exudative (PSE) meat
in poultry can be prevented. Li et al. (2017a, b, c) studied the effect of AMPK on
protein acetylation and glycolysis in postmortem muscles and reported that AMPK
controlled the glycolysis through protein acetylation in pre-slaughter stressed ani-
mals, whereas Mora et al. (2015) derived 68 peptides from ubiquitin-60S ribosomal
protein which contributed to the better knowledge of protein degradation and the
responsible intrinsic enzymes and suggested that these peptides can be used as
1.3 Current Research of Protein Phosphorylation (Live and Postmortem) 5
biomarkers to track the processing time. In postmortem muscles, ubiquitination,

directly related to tenderness, can be an indicator of the extent and rate of meat
tenderization (Picard and Gagaoua 2017). Recently, it was also speculated that nitric
oxide and protein S-nitrosylation were also involved in the conversion of muscle into
meat by regulating the biochemical processes including glycolysis, release of cal-
cium, and breakdown of myofibrillar proteins (Liu et al. 2018).
1.3 Current Research of Protein Phosphorylation (Live

and Postmortem)
Among all post-translation modifications, protein phosphorylation is one of the most

important and common modifications which has been extensively investigated.
Protein phosphorylation is the most important cellular regulatory event, as so
many enzymes and receptors are regulated through the phosphorylation and dephos-
phorylation, and it controls the protein synthesis, cell growth and division, signal
transduction, and process of aging. Phosphorylation is a reversible mechanism,
which is mainly activated by the actions of kinases. During the process, the phos-
phate group is added to the polar side of several amino acids to modify the structure
and allow them to interact with other molecules. On the contrary, dephosphorylation,
mediated by phosphatases, antagonizes the actions of kinases. Different internet
websites, including PhosphoSitePlus (www.phosphosite.org) and PhosphoNET
(www.phosphonet.ca), have listed around 200,000 known phosphosites.
Besides controlling the live animal processes, phosphorylation also has a signif-
icant influence on postmortem muscle biochemistry. By using proteomic tools, many
studies have explored the role of protein phosphorylation in postmortem meat
quality development. Meat color and tenderness, which are the most common
criteria used to predict the meat quality, are affected by protein phosphorylation.
The global phosphorylation level of tough meat is higher than tender meat. Mean-
while, the phosphorylation status of actin and myosin light chain 2 is also critical in
determining the ultimate meat tenderness (Chen et al. 2016; Li et al. 2017a). The
degradation of myofibrillar proteins by μ-calpain, which tenderizes the meat, is also
decreased by its phosphorylation (Li et al. 2017a, b, c). Furthermore, dephosphor-
ylation and protein kinase A (PKA) phosphorylation positively regulate the func-
tions of μ-calpain (Du et al. 2018). Similarly, protein phosphorylation also
significantly affects the meat color stability, by regulating the glycolysis and redox
form of myoglobin (Li et al. 2017c, 2018). Moreover, different patterns of sarco-
plasmic proteins phosphorylation, including pyruvate kinase and triosephosphate
isomerase-1, are related to the postmortem muscle pH decline (Huang et al. 2011).
Season also significantly affects the phosphorylation level of post-slaughter glyco-
lytic enzymes and plays its role in mediating the effects of pre-slaughter environ-
mental temperature on post-slaughter glycolysis and meat quality development
(Li et al. 2015a). It was also reported that electrical stimulation affected the
6 1 Introduction
postmortem rate of pH decline by regulating the phosphorylation of sarcoplasmic

and myofibrillar proteins (Li et al. 2015b).
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Part I
Relationship Between Protein
Phosphorylation and Meat Quality
Chapter 2
Protein Phosphorylation Detection Method
Abstract With the advancement of technical approaches, signaling pathways have

been continuously discovered in numerous biological processes. Majority of these
signaling pathways include the protein phosphorylation, which is a main post-
translational modification affecting the structure and function of proteins within
the cell. Consequently, determining the locations and extent of the protein phos-
phorylation of any specific protein or set of proteins, either qualitatively or quanti-
tatively, has become a routine and extremely vital step in many fields of life sciences,
and recently in meat science as well. However, when choosing the method to detect
protein phosphorylation, it can be different depending on several aspects, particu-
larly under consideration of the availability of the related reagents and equipment.
Although different methods have their own advantages, but this is also a matter of
fact that every method is associated with several drawbacks such as poor specificity,
low sensitivity, safety issues, or intensive labor. In this chapter, we outlined the
routine methods used to detect the protein phosphorylation and discussed their
advantages and disadvantages, especially focusing on the usage of gel and liquid
chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques
to identify phosphorylated proteins in meat science field.
Keywords Protein phosphorylation · Methods · 2DE-gel · Pro-Q Diamond · TiO2

enrichment · LC-MS/MS
2.1 Introduction
Proteins can be modified through several modifications with several functional

groups, such as phosphate, ubiquitin, nitric oxide, sulfate, glycan, ribose, lipid, etc.
Protein phosphorylation involving the phosphate group is an important post-
translational modification that affects the structure and function of proteins. Kinases
can add phosphate group to threonine, serine, or tyrosine residues. On the contrary,
phosphatases have the ability to remove phosphate group from these residues. These
amino acids enclose a nucleophilic group that can react with the adenosine triphos-
phate (ATP), replace the oxygen present on terminal phosphorous, and eject the

https://doi.org/10.1007/978-981-15-9441-0_2
12 2 Protein Phosphorylation Detection Method
adenosine diphosphate (ADP) from the amino acid. In eukaryotic cells, kinases and
phosphatases are mainly in their active form, which makes phosphorylation a vital
post-translational modification for biological processes. It is estimated that 30% of
eukaryotic cell proteins are involved with phosphorylation, which makes it the most
interesting protein modification for the scientists to explore its many biological
functions (Scorsone et al. 1987).
In the past decade, significance of protein phosphorylation in medical field has
urged the meat scientists to study the protein phosphorylation of postmortem
muscles in order to explore the mechanisms of meat quality formation. In this
context, different techniques have been used in this field. However, the principal
approaches to evaluate the protein phosphorylation are gel-based methods to stain
specific phosphoproteins, for instance, Pro-Q Diamond staining (Steinberg et al.
2003). In comparison with traditional antibody-based and radioactive labeling
approaches, Pro-Q Diamond dye has significant benefits such as evasion of radio-
activity, no sequence specific binding to phosphorylated amino acids, and no need of
time-consuming, electro-blotting, or expensive antibodies. Pro-Q Diamond dye
allows the rapid determination of phosphor-serine, phosphor-threonine, and
phosphor-tyrosine proteins, as well as the identification of the phosphoproteins
and their phosphorylated peptides by mass spectrometry (Schulenberg et al. 2003).
In the same way, mass spectrometry is considered as a major tool to study protein
post-translational modifications on large scale, and it also helps to explore the unique
phosphorylated proteins and phosphorylation sites (Mann et al. 2002).
The increase in demand and improvement in methodologies bring researchers to a
closer understanding of the complex processes, which ultimately control cellular
function and meat quality formation. However, to select the best method under
different circumstances, it is important to pick the method that best fits the objective
and experimental design of the study. This chapter will provide a brief overview of
the most commonly used techniques in field of meat science, with special focus of
different gels and LC-MS/MS related techniques to measure protein
phosphorylation.
2.2 Principle of Phosphorylated Protein Detection
Analysis of the phosphorylated protein is necessary for interpreting the fundamental

biological processes and signaling networks at the molecular level. Several methods
have been described for the analysis of phosphorylated proteins according to differ-
ent principles. However, there are several limitations with those methods, so that
none of them could be used to detect all kinds of protein phosphorylation.
One of the methods that used at early stage for detecting protein phosphorylation
is the labeling of proteins with the radiolabeled 32P-orthophosphate (Erikson and
Erikson 1980). In this method, kinase transfers radiolabel to its substrate that allows
to detect its activity. For which, the sample is incubated with the radiolabel for a set
amount of time prior to harvesting and preparing the sample, and isolate the proteins
2.2 Principle of Phosphorylated Protein Detection 13
by gel electrophoresis ( an immunoprecipitation step for a particular protein). Then

make the gel dry and expose it to film on a phosphorimager screen to detect labeled
bands. Radiolabeling works well if we want to determine the protein phosphoryla-
tion without knowing the site of phosphorylation or the kinase involved.
On the other hand, antibodies have turned out to be a great tool to detect protein
phosphorylation at a particular site. Antibodies can be fixed to specific site, thus
identifying related protein phosphorylation. The antibodies are named as phospho-
specific antibodies and are becoming a critical method to identify protein phosphor-
ylation at basic research and commercial diagnosis levels.
Post-translational modifications including protein phosphorylation can be easily
identified on two-dimensional electrophoresis (2DE) gels. The phosphorylation
replaces neutral hydroxyl groups on threonines, serines, or tyrosines with negatively
charged phosphates. Therefore, under pH 5.5, the phosphates added a single nega-
tive charge and adjacent to pH 6.5, phosphates added the 1.5 negative charges,
moreover, beyond pH 7.5, phosphates added 2 negative charges. Thus, relative
amount of each isoform including protein phosphorylation can be easily and rapidly
determined from staining intensity on 2DE gels.
It is possible to detect phosphorylation based on the shift of protein electropho-
retic mobility just on simple 1-dimensional SDS-PAGE gels. SDS-PAGE is a very
versatile technique to separate complex protein mixture and is the most commonly
and widely used technique in different research areas. Radioactive labeling of
phosphorylated proteins combined with gel separation is a very sensitive and
quantitative approach to separate the phosphorylated from the non-phosphorylated
proteins. Detection of antibodies bonded with specific protein or phosphorylated
amino acid could be used to analyze small-scale protein phosphorylation. A major
limitation of this technique is the lack of specificity of the antibody in binding with
specific phosphorylated protein. Another commonly used method is the application
of commercially available Pro-Q Diamond phosphoprotein stain. This is a simple
technique that selectively marks the phosphoproteins of phosphorylated spots on
tyrosine, serine, or threonine residues.
In summary, all the electrochemical methods to detect protein phosphorylation
including SDS-PAGE are usually based on one of the following two principles:
(1) the addition of negative charges to the protein with the transfer of phosphoryl
groups; (2) the release of protons in the reaction buffer upon phosphorylated protein
(Bhalla et al. 2014). There have been also attempts to detect the changes in the
protein charge after phosphorylation by measuring the alterations on the surface
charge of an electrode in contact with the protein, which is recorded in the form of
current as a function of time.
2.3 Gel-Based Phosphorylated Protein Detecting Methods
2.3.1 Separation of Proteins by 2DE Gel
Two-dimensional electrophoresis of proteins, firstly introduced by O’Farrell (1975)

and Klose (1975), was the sign of beginning of proteomics. The 2DE is a commonly
used technique to study the innovative protein mixes extracted from the cell, tissue,
or any other biological sample. According to the name, this technique is mainly
comprised of two steps; isoelectric focusing as first dimension, which separates the
proteins based on their isoelectric point by immobilized pH gradient; and
SDS-PAGE as second dimension, which segregates the proteins based on their
molecular weight (Fig. 2.1). On the two-dimensional array, every spot represents
one kind of protein specie of the tested sample. Then targeted spots are removed and
digested to identify the proteins. Thus, thousands of proteins could be separated and
obtained from the sample by the two mentioned physical techniques of this method.
Although it is an old and no longer the only method of proteomics, it still has
many salient features and advantages. Using 2DE, thousands of proteins can be
identified by a single run, and resolved the intact full length protein on single gel.
The method of 2DE includes the visualized detection of isoelectric point and
molecular weight by quantification based on the spot intensity. Moreover, 2DE is
a compatible technique for further analysis of proteins with many other biochemical
methods, such as polypeptides can also be further identified with antibodies and
tested for post-translational modifications like protein phosphorylation. Further-
more, continuous improvement in its methodologies also increases the robustness
of 2DE. The possible limitations of these techniques are the big amount of sample
handling, smaller dynamic with limited reproducibility. Some of the proteins, with
Fig. 2.1 Images of 2DE gels of the differentially phosphorylated proteins at 4 h postmortem
between groups with different tenderness. The figures on the image and in the parenthesis represent
spot number (Li et al. 2017)
2.3 Gel-Based Phosphorylated Protein Detecting Methods 15
low quantity, acidic, basic, or hydrophobic behavior, of very small or very large size,
are difficult to be separated through this technique.
2.3.2 Detection of Phosphorylated Protein by Pro-Q Diamond

Gel Stain
Fluorescent stains are developed recently to identify the proteins, including their
post-translational modifications, with high sensitivity and linearity. Among these
stains, Pro-Q Diamond phosphoprotein stain is a frequently used technique, com-
mercially manufactured by the Invitrogen™. Pro-Q Diamond is an advanced method
that offers a simple and direct way to bind with the phosphate moiety of phospho-
proteins. It can work without involvement of numerous steps or sample pretreatment
for staining the phosphoproteins, which phosphorylated on threonine tyrosine, or
serine residues. Pro-Q Diamond is compatible with standard SDS-PAGE or with
2DE gels, and there is no need of specific antibody or Western blotting (Fig. 2.2).
This technique can also be combined with mass spectrometry, therefore, allowing
meaningful examination of phosphorylation status of entire proteome for the first
time.
Although the specificity and sensitivity of Pro-Q Diamond gel stain are good,
however, high cost hinders its usage to high-throughput phosphoproteomics. In this
context, recently Wang et al. (2014) introduced an alternative quercetin stain, which
could selectively detect the 16–32 ng of phosphoproteins. Quercetin stain requires
relatively lower cost and readily available chemicals and is more time saving.
2.3.3 Detection of Phosphorylated Protein by Phos-Tag

Method
A dinuclear metal complex of 1, 3-bis[bis(pyridin-2-ylmethyl) amino] propan-2-

olate is a phosphate-binding tag (Phos-tag) aqueous solution, firstly described by
Kinoshita-Kikuta et al. (2007). Manganese homolog (Mn2+-Phos-tag) can be bonded
with phosphate entity, such as phosphotyrosine and phosphoserine at the alkaline pH
(pH ¼ 9.0). The Mn2+-Phos-tag can be polymerized with the separating gel of
SDS-PAGE and used for discovering phosphoproteins. This method does not
involve with radioactive or other chemical labels and phosphate-binding specificity
is also independent from the amino acid sequence, therefore, it is widely used for
determining the phosphorylation state of many proteins.
Phos-tag method had certain constraint in finding the mobility shift with phos-
phorylated forms of some of the proteins. Some proteins treated with different
protein kinases failed to present any change in the band shift using this technique.
16
Fig. 2.2 Images of gels stained with Pro-Q Diamond. Bands marked with * were selected for protein identification by LC-MS/MS. High, moderate, and low
represent high color stability group, moderate color stability group, and low color stability group, respectively. Adapted from (Li et al. 2018)
2 Protein Phosphorylation Detection Method
2.4 Detection of Phosphorylated Proteins by LC-MS/MS 17
Moreover, the alkaline buffer used for the SDS-PAGE gel gave short-term stability
for the separated phosphoproteins by this method.
To deal with limitations of Mn2+-Phos-tag assay, Kinoshita-Kikuta et al. (2007)
modified the protocol and introduced Zn2+-Phos-tag SDS-PAGE. This new method
used the neutral pH buffer and Zinc (II) complex (Zn2+-Phos-tag acrylamide) instead
of using alkaline pH and Mn2+ of the Mn2+-Phos-tag assay. The application of
neutral pH was the main development and it made it possible to store the gels for
nearly 6 months without any deterioration compared to alkaline pH. Additionally,
use of Zn2+-Phos-tag gave a better mobility shift for some of the proteins which
failed to show the shift or showed the smaller shift on the gel with Mn2+-Phos-tag
assay.
2.4 Detection of Phosphorylated Proteins by LC-MS/MS
2.4.1 Labeling Treatment of Samples
Over the past few years, several approaches have been developed for the incorpo-
ration of specific tags into the peptides or proteins. These mainly include the
introduction of isotopic or chemical tags at specific function group on polypeptides,
use of heavy amino acids for metabolic isotope labeling and different methods of
introducing the stable isotopes by enzymatic reactions. Every method has its own
specific strengths and weaknesses. For example, incorporation of stable isotopes by
chemical reactions provides a good selectivity and specificity for tagging the reactive
groups on proteins or peptides. Because it occurs after isolation, avoiding possible
side reactions is important for application of this procedure. While in metabolic
isotope labeling, cell culturing without any chemical reaction is used to label the
proteins, so it is easy to perform. However, this technique is limited to in vitro
labeling of cells through cell culturing. Incorporation of stable isotopes by enzymatic
reactions is straightforward, however, difference created by this technique is some-
times 4 or 2 Da, which is difficult for quantitative analysis without using the
algorithm for deconvolution of isotope pattern.
Among these techniques, isotope coded affinity tag (ICAT) is the most commonly
used method, where proteome of two samples are labeled on the side chains of
reduced cysteinyl residues using one chemically same but isotopically different
reagent. Important feature of this technique is the introduction of biotin loving tag
in the ICAT reagents, which helps in selective isolation and purification of analytes,
thus enabling a substantial reduction in sample complexity.
2.4.2 Label Free Treatment of Samples
Label free treatment of samples for LC-MS/MS is a smart method for high-
throughput quantitative proteomics that aims to detect the relative abundance of
proteins of two or more biological samples. Unlike other techniques, label free
quantification does not use a stable isotopes and is comprised of two main
approaches: (1) measurement of peptide peak intensity, which measures and com-
pares the peaks of chromatographic peptide precursors ions of a particular proteins;
and (2) spectral counting, which involves the calculating and matching of the
number of spectra of the tested protein peptides in order to know the relative quantity
of that specific protein. Both of these approaches are simple and economical with
high reproducibility at both protein and peptide levels. However, application of these
procedures requires more computational power accomplished with the handling of
chromatographic peaks, peptide intensity measurement, and spectral counting for
examining the small significant changes, such as protein phosphorylation, that are
biologically meaningful. The main steps involved in both mentioned procedures are:
(1) preparation of sample involving extraction, reduction, alkylation, and digestion
of proteins; (2) sample separation and analysis by LC-MS/MS; (3) data analysis
including identification, quantification, and statistical analysis; and at the end
(4) interpretation of results.
In recent time, an increasing number of publications show that label free quan-
tification is a viable and appropriate method to identify biomarkers within given
samples. Besides its breakthrough in biological sciences, there are several issues
regarding this method. These issues mainly include the problems related with
chromatographic alignment, peptide qualification for quantification and normaliza-
tion. However, advancements in label free protein quantification methods will
further assist to fulfill the demands of advanced proteomics.
2.4.3 Enrichment of Phosphorylated Peptides Using

the TiO2 Beads
Since past few years, adsorption of proteins with titanium dioxide (TiO2) has been
explored extensively with the goal of finding a technique for studying the
bioelectrochemical functions of proteins. Interestingly for the study of
phosphoproteomics, TiO2 is shown to have affinity with phosphate ions, and TiO2
chromatography is used as an effective method for phosphopeptides enrichment.
Several scientists introduced the enrichment of phosphorylated peptides using the
TiO2 material. For example, Pinkse et al. (2004) explored the capability of TiO2 to
bind with the selective phosphopeptides by using the online two-dimensional LCMS
setup with the particles of TiO2 as the first dimension and reversed phase material as
second dimension. Thingholm et al. (2006) introduced another offline setup TiO2
chromatography with stronger buffers, including the use of 2, 5-dihydrobenzoic acid
2.5 Conclusions 19
and comparatively higher concentrations of trifluoroacetic acid, which reduced the

non-specific binding of TiO2 with compounds other than phosphorylated peptides.
The higher selectivity of TiO2 with the phosphopeptides makes it a stronger
method for phosphoproteomics study. Furthermore, this technique is exceptionally
acceptable towards most of the salts and buffers used in biochemistry and cell
biology studies. Because the whole procedure only requires approximately 15 min
per sample with prepared buffers, the application of the TiO2 beads for enrichment of
phosphorylated peptides is also a highly robust technique and has become the first
choice in laboratory as well as in large-scale phosphoproteomic studies. Another
benefit of this technique is the highly efficiency of phosphopeptides purification,
which is good to study both in vitro and in vivo phosphoproteins when combined
with mass spectrometry.
2.4.4 LC-MS/MS Analysis
Mass spectrometry (MS) is the most modern approach for the determination of
phosphorylation and it significantly advances the research in protein phosphoryla-
tion. MS is not only used for detection of phosphorylation but also helps to identify
the phosphorylation sites. Determination of phosphorylation by mass spectrometry
is based on the spectrum generated by the peptides which are previously digested by
trypsin. Liquid chromatography with tandem mass spectrometry is the most advance
form of MS and has been extensively used for phosphoproteomic studies as a
powerful analytical approach that uses the separating ability of liquid chromatogra-
phy along with the enhanced sensitivity and selectivity MS capability of triple
quadrupole MS.
The LC-MS/MS has many advantages over other phosphoproteomic techniques,
as liquid chromatography offers a vast range of separation options. It provides high
specificity detectors and there is no need for confirmatory detection. Similarly, it also
shows high sensitivity and can identify the compounds lower than 1 part per trillion.
LC-MS/MS has great reproducibility if internal standards are stable labeled. It also
has restrictions, such as, when the internal standards are not stable labeled, the
reproducibility is usually not as good as other LC detectors. It is also limited for
large molecules (>4000 m/z), and the matrix co-extractants can suppress or boost
the ionization potential and may needed to apply matrix matched calibrants for
quantification.
2.5 Conclusions
Protein phosphorylation is an extraordinarily important component of various signal

transduction pathways underlying postmortem biochemical processes. In this chap-
ter, we summarized the different approaches commonly used in meat science field.
However, despite all these given and other available techniques, detection and
quantification of protein phosphorylation in any given biological samples is still
hampered and challenging. The study of protein phosphorylation on a large scale has
been limited due to intensive labor, high time consuming, and low cost effective.
Therefore, it is urgently needed to discover the simple, reliable, and rapid techniques
for wider application of protein phosphorylation to effectively explore the hidden
part of meat quality formation.
Acknowledgments Parts of this chapter are reprinted from Journal of the Science of Food and
Agriculture, 97, Li, X., et al., A comparative analysis of phosphoproteome in ovine muscle at early
postmortem in relationship to tenderness, 4571-4579; Food Chemistry, 240, Li, M., et al., Com-
parative profiling of sarcoplasmic phosphoproteins in ovine muscle with different color stability,
104-111. Copyright (2020), with permission from Elsevier.
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Chapter 3
Protein Phosphorylation Affects Meat Color
Abstract The relationship between meat color stability and protein phosphorylation
was studied. By adding phosphatase and protein kinase inhibitors into sarcoplasmic
proteins from minced ovine longissimus thoracis et lumborum (LTL) muscle, we
found out that protein phosphorylation had a significant relationship with lactate
accumulation, as well as pH decline rate and extent. Also, meat color stability had a
negative correlation with sarcoplasmic protein phosphorylation level. Therefore,
how protein phosphorylation regulated glycolysis and myoglobin redox forms, and
furthermore meat color stability was investigated in the second step. Results showed
that phosphorylation differences existed in some individual protein bands, but not in
whole sarcoplasmic proteins, from muscle groups with different meat color stability.
Glycolytic enzymes turned out to be the most color stability related proteins, and
myoglobin phosphorylation level was negatively related to meat color stability,
which meant protein phosphorylation might regulate meat color stability via glycol-
ysis and myoglobin redox stability. At the third step, the protein phosphorylation
level of muscles with different color stability was examined with quantitative
analysis. We successfully identified 3412 phosphopeptides from 1070 phosphopro-
teins, and among which 243 had a significant correlation between phosphorylation
level and meat color stability. With further study into these phosphopeptides through
informatics analysis, 27 of them were highly related to meat color stability, and
glycolytic enzymes were the biggest group. Moreover, there was a negative corre-
lation between the Ser133 phosphorylation level in myoglobin and meat color
stability. In conclusion, our results revealed that protein phosphorylation may
regulate meat color stability through glycolytic enzymes and myoglobin redox
forms, and Ser133 played a key role in the process.
Keywords Meat color · Protein phosphorylation · Sarcoplasmic proteins ·

Glycolysis · Glycolytic enzyme · Phosphoproteomics
Electronic Supplementary Material The online version of this chapter (https://doi.org/10.1007/

978-981-15-9441-0_3) contains supplementary material, which is available to authorized users.

https://doi.org/10.1007/978-981-15-9441-0_3
24 3 Protein Phosphorylation Affects Meat Color
3.1 Introduction
With the improvement of residents’ living standards, consumers’ demand for food
has changed from quantity to quality, safety, and nutrition. Meat and meat products
are indispensable parts of human diet. The evaluation indexes of meat quality mainly
include tenderness, flavor, meat color, water resistance, and juiciness. Among them,
meat color is the most intuitive impression of consumers when they purchase meat or
meat products (Yin et al. 2011). Meat and meat products with poor color give
consumers the impression of unsanitary and low quality, resulting in huge economic
losses caused by the discount sale of meat due to the deterioration of meat color
every year (Jeyamkondan et al. 2000; Canto et al. 2015). Therefore, meat color is one
of the most important factors influencing consumers’ purchase decisions (Clydes-
dale 1978; Mancini and Hunt 2005).
Protein phosphorylation is one of the most common and important post-
translational modifications in biology system. It refers to the process of transferring
γ-phosphorylation group from GTP or ATP to the amino acid substrate protein
residue under the catalysis of kinase, which mainly occurs on the side chains of
serine, tyrosine, and threonine. Phosphatase can dephosphorylate proteins, which is
a dynamic balance, and the result of its comprehensive action determines the
phosphorylation state of proteins (Fischer 1993; Krebs 1993). The phosphorylation
and dephosphorylation of proteins play an important role in the process of cell signal
transmission, as a switch that controls many cell biological functions, which involve
almost all vital activities, including cell signal transduction, gene expression, apo-
ptosis, muscle contraction, metabolism, tumor generation, etc. (Cohen 2002).
3.2 Changes of Meat Color After Protein Phosphorylation

Regulation
Protein phosphorylation in postmortem muscle played essential roles in meat qual-

ity. The influence of protein phosphorylation on meat color stability was investigated
in the minced ovine longissimus thoracis et lumborum. After trimming off visible fat
and connective tissue, the LTL muscle was divided into four treatment groups (150 g
for each group): phosphatase inhibitor group (high phosphorylation level), saline
control group (C1), muscle was added with the vehicle of saline, kinase inhibitor
group (low phosphorylation level) and DMSO control group (C2), muscle was
added with dimethyl sulfoxide (DMSO) and saline. The lactate and pH values
were measured to check the protein phosphorylation while the protein band was
studied to evaluate the intensity of phosphoproteins. Moreover, the analysis of meat
color and the relative content of myoglobin redox forms calculations were used to
check the meat color stability related to the phosphorylation of sarcoplasmic pro-
teins. Thus, the protein phosphorylation may be involved in meat color development
by regulating glycolysis and the redox stability of myoglobin.
3.2 Changes of Meat Color After Protein Phosphorylation Regulation 25
3.2.1 Global Level of Protein Phosphorylation After

Regulation of Protein Phosphorylation
Gel-based analysis of sarcoplasmic protein phosphorylation was shown in Fig. 3.1

and Table 3.1. As expected, the overall phosphorylation of sarcoplasmic proteins in
the minced meat with phosphatase inhibitor was significantly higher (P < 0.05) than
in control and kinase inhibitor added samples. In addition, the global phosphoryla-
tion of sarcoplasmic proteins in the kinase inhibitor group became even lower
(P < 0.05) than in both control samples.
No difference in protein phosphorylation was detected between the two control
samples, which were intermediate among the four treatments. This result confirmed
that the overall phosphorylation of sarcoplasmic proteins was altered by inhibitors.
Fig. 3.1 Gel-based analysis of sarcoplasmic protein phosphorylation. (a) Image of gels stained
with Pro-Q Diamond. (b) Image of gels stained with SYPRO Ruby. P, C1, C2, and K represent
phosphatase inhibition group, saline control group, DMSO control group, and kinase inhibition
group, respectively (Li et al. 2017)
Table 3.1 Effects of protein kinase and phosphatase inhibitors on the global phosphorylation of
sarcoplasmic proteins (Li et al. 2017)
Group
Display time Phosphatase Kinase
(hours) inhibition Saline control DMSO control inhibition
2 0.97 0.002aA 0.80 0.013bA 0.80 0.014bA 0.71 0.015bA
24 1.00 0.009aA 0.84 0.008bA 0.81 0.006bA 0.65 0.011cB
48 1.11 0.011aB 0.83 0.030bA 0.78 0.004bA 0.54 0.008bC
72 1.18 0.002aBC 0.79 0.019bA 0.83 0.016abA 0.54 0.004bC
120 1.17 0.002aBC 0.87 0.011bA 0.84 0.009bA 0.62 0.008bB
168 1.21 0.001aC 0.91 0.006aA 0.87 0.001abA 0.67 0.015bAB
Data are expressed as mean standard deviation (n ¼ 5). Means with different lower case letters in
the same row are significantly different (P < 0.05). Means with different upper case letters in the
same column are significantly different (P < 0.05)
3.2.2 Color of Meat with Different Protein

Phosphorylation Level
L* and b* values are presented in Table 3.2. L* values did not differ among the four
treatments at any time points. L* values of the phosphatase inhibition, C1 and C2
groups increased (P < 0.05) for the first 24 h and then kept constant afterwards,
whereas L* values of the kinase inhibition group did not change throughout the
whole experiment period. It has been reported that changes in L* value were subtle
during the experimental period (McKenna et al. 2005). The b* values for all meat
samples were similar to L* values. No significant differences were observed in b*
values among the four groups at all time points except for 168 h. In addition, the b*
values in all samples increased (P < 0.05) for the first 24 h and then kept stable
throughout the rest of the experimental period.
According to Olivera et al. (2013), redness was the most important color param-
eter for fresh meat, which decreased with the increase of storage time. In consistence,
a* values determined in the present study (Table 3.2) kept decreasing during the
whole experimental period. Meat redness was higher (P < 0.05) in kinase inhibition
group than in control C1 and C2 groups, which was greater (P < 0.05) than in
phosphatase inhibition group at all times except for 2 h. No difference in a* values
was observed among the four groups at 2 h. When compared with the other three
groups, a* values in the phosphatase inhibition group declined fastest, which
displayed a rapid decrease in a* values for the first 48 h and the a* values decreased
gradually afterwards. The changes in a* values were similar for the two control
groups. Besides, a* values of the kinase inhibitor added meat declined slowest
(P < 0.05) among the four groups during the 7 days of setting. This implied that
a* values of meat were obviously affected by protein phosphorylation.
Table 3.2 Effects of inhibitors and storage time (hour) on L*values, a*values, b*values (Li et al. 2017)
Display time (hours)
Attribute Group 2 24 48 72 120 168
L*value (lightness) Phosphatase 42.03 3.14aX 44.65 3.80abX 48.53 3.64bX 47.70 3.22bX 48.25 3.68bX 48.49 3.48bX
inhibition
Saline control 43.86 2.61aX 46.33 3.67abX 47.73 2.63abX 48.15 2.82bX 48.81 2.61bX 49.16 2.81bX
DMSO control 41.10 2.71aX 45.25 2.68bX 46.45 1.84bX 46.12 2.61bX 46.47 2.21bX 47.45 2.27bX
Kinase inhibition 42.61 2.44aX 45.37 2.77aX 44.78 2.33aX 44.17 2.88aX 44.51 3.78aX 45.16 2.71aX
a*value (redness) Phosphatase 11.50 1.10aX 8.42 1.21aX 6.95 1.05bX 6.04 0.68bX 5.24 0.68cX 4.96 0.16cX
inhibition
Blank control 12.43 0.56aX 11.28 0.55bY 9.79 0.49 cY 9.47 0.57cdY 8.98 0.42deY 8.65 0.49eY
DMSO control 12.04 0.81aX 10.60 0.87bY 9.63 0.44 cY 9.39 0.45cY 8.94 0.43cdY 8.43 0.71dY
Kinase inhibition 12.33 0.45aX 12.64 1.10bZ 11.38 11.58cZ 11.16 0.12cdZ 9.93 0.21dZ 9.33 0.47dZ
b*value Phosphatase 9.64 1.49aX 11.14 1.00abX 12.48 2.08bcX 14.00 1.57cX 13.72 2.04cX 13.48 0.93cX
(yellowness) inhibition
Saline control 10.02 0.69aX 12.38 0.88bX 11.93 1.54bX 12.30 1.41bX 12.25 1.73bX 12.27 1.22bXY
3.2 Changes of Meat Color After Protein Phosphorylation Regulation
DMSO control 9.24 0.85aX 11.20 0.96bX 11.36 1.19bX 12.08 1.58bX 11.75 1.68bX 11.39 1.55bY
Kinase inhibition 10.06 0.98aX 12.58 1.31bX 12.54 1.14bX 13.44 1.39bX 12.49 0.99bX 12.68 0.92bXY
Data are presented as mean standard deviation (n ¼ 5). Data with different lower case letters in a row are significantly different (P < 0.05). Data with different
capital letters in the same column are significantly different (P < 0.05)
27
3.2.3 Role of pH and Lactic Acid Content on Color of Meat

with Different Protein Phosphorylation Level
The pH values were shown in Fig. 3.2. There was no difference in pH values
between the phosphatase inhibition group and the two control groups. However,
the pH of the kinase inhibition group was significantly higher than those of the other
three groups (P < 0.05) throughout the entire postmortem period except for 2 h
postmortem, at which no difference in pH values was found among all the four
groups. The pH values of phosphatase inhibition group, C1 and C2 control groups
declined to the ultimate values of 5.67, 5.67, and 5.65, respectively, within 24 h, but
pH of kinase inhibition group did not declined to the ultimate value of 6.02 till 78 h.
This meant the pH of the kinase inhibition group declined much slower during the
early postmortem stage (P < 0.05) as compared to the other three groups. The
difference in meat pH values indicated that the rate and extent of pH decline in
postmortem muscle were significantly affected by protein phosphorylation.
Meat quality was largely affected by the rate and extent of pH decline. pH values
in postmortem muscle, which was controlled by the accumulation of lactate and H+
through glycogenolysis and glycolysis (Scheffler and Gerrard 2007), were probably
one of the most important factors affecting meat color. The rate and extent of pH
Phosphatase inhibition Saline control

DMSO control kinase inhibition
6.8
6.6
Aa
6.4
Ab Ab
6.2 Abc Abc
Ac
6.0 Bx
pH
5.8
5.6
5.4 By By By By By
5.2
5.0
2 24 48 72 120 168
Fig. 3.2 pH values of muscles added with or without enzyme inhibitors. Data with different lower
case letters within treatments are significantly different (P < 0.05). Data with different capital letters
at the same time points are significantly different (P < 0.05). Data are presented as mean standard
deviation, n ¼ 5 (Li et al. 2017)
3.2 Changes of Meat Color After Protein Phosphorylation Regulation 29

Lactic acid˄umol/g muscle˅ DMSO control kinase inhibition
140
120 aaa aaa bb
aaa b bb a
b c
100 b a c
aaa
b
80
60
40
20
0
2 24 48 72 120 168
Fig. 3.3 Lactate content in muscles added with or without enzyme inhibitors. Data with different
letters at the same time point are significantly different (P < 0.05). Data are presented as
mean standard deviation, n ¼ 5 (Li et al. 2017)
decline significantly influenced protein characteristics in postmortem muscle, for

example, myoglobin was easier to be oxidized under lower pH conditions and this
reaction was very pH dependent. Gutzke and Trout (2002) reported that pH had a
consistent effect on the myoglobin autoxidation rate for the different species and
temperatures. The effects of pH on the oxidation of myoglobin have been studied
from pH 4.8 to pH 12.6, and the result turned out that this autoxidation reaction
depended on hydrogen ion concentration directly (Shikama and Sugawara 1978). It
is logical to conclude that the lower rate and extent of pH decline were one of the
reasons for the higher color stability in kinase inhibition groups comparing to the
other three groups. Although both of the phosphorylation level and color stability
differ significantly, no difference was found in pH value between phosphatase
inhibition group and control groups. This implied that there may be some other
reasons for the influence of protein phosphorylation on meat color stability except
pH value though the rationale remains unclear.
Lactic acid concentrations in muscle corresponded well with the pH values. No
difference was observed in lactic acid content (Fig. 3.3) between C1 and C2 control
groups. In addition, no difference in lactic acid was detected between phosphatase
inhibition group and the two control groups at most time points, but the kinase
inhibition group had lower lactic acid content than the other three groups (P < 0.05)
throughout the entire experimental period. This result further supported that muscle
with lower protein phosphorylation had lower (P < 0.05) glycolytic rate when
compared with samples with higher protein phosphorylation. Consequently, it was
proposed that protein phosphorylation had an influence on the rate and extent of pH
decline through regulating glycolysis in postmortem muscle.
Most of the glycometabolic enzymes can be phosphorylated, such as glycogen
phosphorylase (GP), pyruvate kinase (PK), and phosphofructokinase (PFK), which
were glycometabolic rate-limiting enzymes. Protein phosphorylation was the most
common post-translational modifications, which was a key modulator of protein
structure, function, and activity. Several previous studies have revealed that protein
phosphorylation regulates the activity of those glycometabolic rate-limiting
enzymes. For example, after phosphorylation, PK was transformed to a more acid-
stable isoform, and maintained high activity in PSE meat (Schwägele et al. 1996).
GP can be phosphorylated on serine 14, leading to changes in its structure and
activation of its activity (Johnson 1992; Sprang et al. 1988). Phosphorylation of PFK
regulated the compartmentalization of the enzyme to provide energy to the cellular
component by forming a complex with actin (Cai et al. 1997; Kuo et al. 1986).
Protein phosphorylation regulated postmortem glycolysis by regulating the activity
or stability of glycolytic enzymes (Huang et al. 2011). Shen and Du (2005) reported
that the glycolysis and pH decline were indirectly affected by the phosphorylation
status of AMP-activated protein kinase (AMPK) in postmortem muscle. In brief, it
can be concluded that protein phosphorylation regulated the rate and extent of pH
decline in postmortem muscle by controlling the glycolysis reactions. Since protein
phosphorylation altered the structure, activity, and stability of protein, it was likely
that phosphorylation of myoglobin influenced meat color stability by regulating its
redox stability. However, the phosphorylation of myoglobin was not measured in the
present study. Therefore, whether the phosphorylation of myoglobin affected color
stability was uncertain, and further research was needed.
3.3 Pattern of Sarcoplasmic Protein Phosphorylation

in Meat with Different Color Stability
Protein phosphorylation might have impact on the regulation of meat color stability
probably by regulating glycolysis and the redox stability of myoglobin. The phos-
phorylation of sarcoplasmic proteins in postmortem muscles was investigated in
relationship to color stability in the present study. The longissimus thoracis et
lumborum muscles were used in the study. Each side of the carcasses was fabricated
into six steaks, individually wrapped in an oxygen-permeable polyvinylchloride
(PVC) film, and stored at 4 C for 8 days to simulate retail display. The first steak
from the left side was used for pH measurement at 45 min and 24 h postmortem. The
second steak was used for color determination. The rest of the steaks were used for
muscle sample collection at 45 min, 6 h, 24 h, 2 days, 3 days, 4 days, 5 days, 6 days,
7 days, and 8 days postmortem. The meat color parameters were studied to check the
color stability in the meat. Liquid chromatography—tandem mass spectrometry
(LC-MS/MS) was used for identification of phosphoproteins. The gel
3.3 Pattern of Sarcoplasmic Protein Phosphorylation in Meat with Different Color. . . 31
electrophoresis was used for the separation of protein and protein phosphorylation
level determination. To better understand the biochemistry of meat color stability,
protein phosphorylation was comparatively profiled in muscles with different color
stability.
3.3.1 Global Sarcoplasmic Protein Phosphorylation Level

of Meat with Different Color Stability
Phosphoproteins were detected by SDS-PAGE and fluorescence staining. As shown

in Fig. 3.4, a total of 24 individual proteins bands were detected on the SDS-PAGE
gels and the intensity of phosphorylated protein to total protein ratios (P/T ratios) of
these protein bands was calculated (Fig. 3.4). No significant difference in global
phosphorylation level of sarcoplasmic proteins was detected among the three groups
during the whole display period.
However, the total phosphorylation level of sarcoplasmic proteins varied with
time in all three groups, which decreased (P < 0.05) within the first 24 h and then did
not change afterwards.
High Moderate Low

Phosphorylation level˄P/T
1.1 a a
a ab
1.0 ab abc
ab b b
bc bc bc
0.9 b b c bc c
c
ratio˅
0.8
0.7
0.6
0.5
45min 6h 24h 3d 5d 7d
Display time
Fig. 3.4 Calculated global phosphorylation levels of sarcoplasmic proteins. High, moderate, and
low represent high color stability group, moderate color stability group, and low color stability
group, respectively. Data are expressed as mean standard deviation (n ¼ 5). Values with different
letters are significantly different within the same group at different display time (P < 0.05) (Li et al.
2018a)
3.3.2 Correlation of Meat Color Parameters

and Sarcoplasmic Protein Phosphorylation Level
All the 24 protein bands were subjected to association analysis (Table 3.3). Pearson
correlation coefficients between meat color attributes and the total phosphorylation
level of sarcoplasmic proteins or the phosphorylation levels of the 24 individual
protein bands were shown in Table 3.3. Statistical analysis showed that the total
phosphorylation level of sarcoplasmic proteins negatively correlated (P < 0.01) with
meat L (r ¼ 0.897), a (r ¼ 0.738), b (r ¼ 0.848) values and the relative
content of oxymyoglobin (r ¼ 0.816), but positively correlated (P < 0.01) with the
relative content of DeoxyMb (r ¼ 0.885).
This suggested that meat color stability was correlated with the total phosphor-
ylation level of sarcoplasmic proteins, which was in accordance with our previous
study (Li et al. 2017). The results of Pearson’s correlation analysis between the
phosphorylation levels of individual protein bands and L, a, b, R630/580 values
and the relative content of deoxymyoglobin, oxymyoglobin, and metmyoglobin
were shown in Table 3.3. A total of 11 protein bands were found to correlate with
instrumental color values, which were band 5, 6, 8, 9, 11, 12, 13, 14, 15, 20, and 23.
The P/T ratios of these bands were presented in Fig. 3.5.
In several previous studies, the differential proteome was investigated in rela-
tionship to meat color stability. Canto et al. (2015) reported that the expression/
abundance of some sarcoplasmic proteins might influence meat color stability. They
reported that phosphoglucomutase-1, glyceraldehyde-3-phosphate dehydrogenase,
pyruvate kinase M2, and creatine kinase M-type were over-abundant in color-stable
steaks, while adenylate kinase isoenzyme 1 and myoglobin were overabundant in
color-labile samples. A study by Wu et al. (2015) showed that fructose-bisphosphate
aldolase A isoform, pyruvate kinase isozyme M1/M2 isoform, and malate dehydro-
genase were negatively correlated, while L-lactate dehydrogenase A chain isoform
was positively correlated with beef a values and metmyoglobin reductase activity
(MRA). Joseph et al. (2012) reported that creatine kinase and β-enolase had a
positive correlation with meat a value. In a study by Gao et al. (2016), it was
reported that creatine kinase M-type, malate dehydrogenase, and β-enolase were
related to meat color development. Based on all these studies, it can be concluded
that proteome played a role in meat color development and discoloration, though the
underlying mechanism was not completely understood. The color-related proteins
discussed above were also identified in the present study. Most of these proteins are
glycolytic enzymes that can be reversibly phosphorylated, such as phosphogluco-
mutase 1 (PGM1), PK, fructose-bisphosphate aldolase A (ALDOA), glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDHA), and
β-enolase (Huang et al. 2011; Kachel et al. 2015). Phosphorylation regulated enzyme
activity, stability, and interaction with other proteins (Sale et al. 1987). Several
studies have revealed that protein phosphorylation played key roles in regulating
the activities of most glycolytic enzymes, like pyruvate kinase, which could be
transformed to a more active and acid-stable isoform after phosphorylation
Table 3.3 Pearson correlation coefficients between meat color attributes and the phosphorylation level of total sarcoplasmic proteins or individual protein
bands (Li et al. 2018a)
L* value a* value b* value R630/580 DeoxyMb OxyMb MetMb
Total phosphorylation level 0.897** 0.738 ** 0.848** 0.046 0.885** 0.816** 0.110
Band1 0.396 0.337 0.462 0.471* 0.534* 0.373 0.700**
Band2 0.252 0.457 0.445 0.138 0.341 0.450 0.065
Band3 0.321 0.180 0.109 0.758** 0.346 0.055 0.689**
Band4 0.489* 0.414 0.497* 0.028 0.577 * 0.433 0.205
Band5 0.664** 0.387 0.624** 0.515* 0.720** 0.509* 0.676**
Band6 0.850** 0.570* 0.798** 0.357 0.885** 0.695** 0.538**
Band7 0.305 0.002 0.122 0.450 0.253 0.082 0.408
Band8 0.787** 0.505* 0.717** 0.323 0.854** 0.604** 0.462
Band9 0.743** 0.580* 0.777** 0.441 0.817** 0.678** 0.718**
Band10 0.295 0.35 0.351 0.051 0.395 0.356 0.190
Band11 0.854** 0.656** 0.821** 0.267 0.872** 0.739** 0.484*
Band12 0.543* 0.078 0.348 0.447 0.564** 0.205 0.387
Band13 0.722** 0.467 0.708** 0.518* 0.775** 0.571* 0.776**
Band14 0.324 0.097 0.261 0.509* 0.402 0.166 0.571*
Band15 0.250 0.191 0.082 0.530* 0.224 0.067 0.390
Band16 0.520* 0.410 0.512* 0.060 0.500* 0.498* 0.182
Band17 0.094 0.113 0.090 0.041 0.051 0.140 0.021
Band18 0.585* 0.338 0.525* 0.495* 0.654** 0.421 0.623**
Band19 0.093 0.170 0.024 0.560* 0.162 0.121 0.578*
Band20 0.391 0.496* 0.383 0.564* 0.356 0.463 0.543*
Band21 0.460
3.3 Pattern of Sarcoplasmic Protein Phosphorylation in Meat with Different Color. . .
0.489 * 0.541* 0.556* 0.029 0.567* 0.179

Band22 0.443 0.340 0.384 0.212 0.328 0.397 0.190
(continued)
33
34
Table 3.3 (continued)

L* value a* value b* value R630/580 DeoxyMb OxyMb MetMb
Band23 0.393 0.340 0.490* 0.358 0.566* 0.385 0.595**
Band24 0.313 0.184 0.314 0.441 0.334 0.240 0.593**
Significant levels: *, 0.01 < P < 0.05; **, P < 0.01
R630/580 was calculated by the ratio of reflectance at 630 to 580 nm as an indicator of meat color stability, DeoxyMb means deoxymyoglobin, OxyMb means
oxymyoglobin, and MetMb means metmyoglobin
3 Protein Phosphorylation Affects Meat Color
2.0 1.5
Band 5 Band 6
a
1.5 b
1.0
P/T ratio
b
P/T ratio
a ab a
1.0 ab
b
b 0.5
0.5
0.0 0.0
45min 6h 24h 3d 5d 7d 45min 6h 24h 3d 5d 7d
Display time Display time
1.5
Band 8 2.0 Band 9
a b
1.0 a aa aa
P/T ratio
1.5
b aab
P/T ratio
b a ab
a a a 1.0 b bab
0.5 bb bb
bb bb
0.5
0.0
45min 6h 24h 3d 5d 7d 0.0
45min 6h 24h 3d 5d 7d
1.5 Band 11 Band 12

1.5
a ab
ab a ab b a ab a b
1.0 1.0 b
P/T ratio
P/T ratio
b
0.5 0.5
0.0 0.0
Fig. 3.5 The phosphorylation levels of 11 color stability related protein bands. High, moderate,
and low represent high color stability group, moderate color stability group, and low color stability
group, respectively. Data are presented as mean standard deviation (n ¼ 5). Different letters at the
same time mean significant difference among groups (P < 0.05) (Li et al. 2018a)
(Schwägele et al. 1996). As phosphorylation regulated the activity and stability of

glycolytic enzymes, the reversible phosphorylation of these color-related proteins
may play a role in the meat color development by regulating the glycolysis. The
main protein identified in band 23 (Fig. 3.1a, b) was myoglobin in the present study,
which was the primarily pigment responsible for meat color. The phosphorylation of
myoglobin has been confirmed in our lab (unpublished) and the phosphorylation
sites of myoglobin of some species are available on the PhosphoSitePlus Web site.
According to the results of one-way analysis of variance (Fig. 3.5, band 23), the
phosphorylation level of myoglobin in the high color stability muscle was signifi-
cantly lower (P < 0.05) than that in the low color stability group within the first 24 h
postmortem. Although no difference in the phosphorylation level of myoglobin
2.0
Band 13 Band 14
ab b a 1.4
1.5 bba 1.2 a ab
a abb
P/T ratio
1.0 b
P/T ratio
1.0 0.8
0.6
0.5 0.4
0.2
0.0 0.0
Band 15 Band 20
2.0
1.5 a
bb a
1.5 bb
P/T ratio
a
P/T ratio
a b ab ab 1.0
1.0 b
0.5
0.5
0.0 0.0
1.5 Band 23
bb c
bb ab
a a
1.0
P/T ratio
0.5
0.0
45min 6h 24h 3d 5d 7d
Display time
High Intermediate Low
Fig. 3.5 (continued)
existed between the moderate and low color stability groups at 45 min and 6 h
postmortem (Fig. 3.5, band 23), higher (P < 0.05) phosphorylation level was
detected in low color stability muscle at 24 h. This result indicated that the phos-
phorylation level of myoglobin was inversely related to meat color stability. In
addition, correlation analysis (Table 3.3) showed that the phosphorylation level of
myoglobin (band 23) was positively correlated with (r ¼ 0.595) the content of
metmyoglobin, suggesting that phosphorylated myoglobin might be more suscepti-
ble to oxidation. As phosphorylation changed the structure and stability of proteins,
it was likely that phosphorylation changed the structure of myoglobin and its
susceptibility to oxidation as observed in the present study (Díaz-Moreno et al.
2009; Zhang and Liu 2017). A recent study has reported that human neuroglobin
underwent hypoxia-dependent phosphorylation (Ascenzi et al. 2013), it was possible
that the difference in myoglobin phosphorylation between treatments was contrib-

uted by the difference in oxygen availability. In summary, the phosphorylation of
myoglobin may play an important role in keeping meat color stable. This was
consistent with a previous study (Canto et al. 2015) which observed an acidic shift
of isoelectric point (pI) of myoglobin on 2-DE gels in greater color lability muscle.
As a pI acidic shift was a symbol for phosphorylation of proteins with pI >6.4 (Zhu
et al. 2005). Thus, phosphorylation modification could lead to this kind of pI shift in
myoglobin, and phosphorylation might play a negative role in the regulation of meat
color stability.
3.3.3 Identification of Phosphorylated Sarcoplasmic Proteins

Related with Meat Color
The eleven interested protein bands were excised from gels and proteins were
identified by LC-MS/MS (Table 3.4). More than one protein was identified in
every protein band. Proteins that had an inappropriate molecular weight or a very
low percentage of coverage of the entire amino acid sequence or a very low score
were excluded. Some proteins were also identified to be presented in more than one
band, which may be caused by proteolytic cleavage of the proteins or protein
polymorphisms. The functions of all identified proteins were annotated using Protein
Knowledgebase (UniProtKB in www.uniprot.org). Most of the identified proteins
were glycolytic enzymes, which included glucose-6-phosphate isomerase (GPI),
fructose-bisphosphate aldolase (ALDOA), phosphoglycerate kinase 1, L-lactate
dehydrogenase A chain (LDHA), alpha-enolase, beta-enolase,
phosphoglucomutase-1 (PGM1), pyruvate kinase (PK), and glyceraldehyde-3 phos-
phate dehydrogenase (GAPDH). Besides, myoglobin, adenylate kinase isoenzyme
1, malate dehydrogenase, creatine kinase M-type, alpha-1, 4 glucan phosphorylase,
sarcoplasmic/ endoplasmic reticulum calcium ATPase 1, calcium-transporting
ATPase, and several other proteins were also identified.
Creatine kinase M-type was identified in both band 12 and band 13 (Table 3.4).
Although no difference was observed in the phosphorylation level of band 12 among
the three groups within the first 24 h PM (Fig. 3.5, band 12), it was lower (P < 0.05)
in the low color stability group than that in the high color stability group on day 3, 5,
and 7 (Fig. 3.5, band 12). Adenylate kinase isoenzyme 1 was the main protein
identified in band 20 (Table 3.4). The phosphorylation level of band 20 was higher
(P < 0.05) in the high color stability group than in the low color stability group at
24 h and 5 days PM (Fig. 3.5, band 20). Besides, the phosphorylation level of band
20 was positively (P < 0.05) correlated with a and R630/580 values, and negatively
(P < 0.05) correlated with the content of metmyoglobin (Table 3.3). The main
proteins identified in band 5 were uncharacterized protein (GN ¼ AKAP4), sarco-
plasmic/endoplasmic reticulum calcium ATPase 1, and calcium-transporting
ATPase (Table 3.4). The main proteins identified in band 6 were uncharacterized
38
Table 3.4 Identified proteins in the 11 color stability related protein bands by LC-MS/MS (Li et al. 2018a)
Band Accession MW Seq.cov
Protein namea nob noa Scorec (Da)d MPe (%)f Organismg Function
Uncharacterized protein (GN ¼ AKAP4) 5 F1MYH5 7300 95,481 4 13 Bos Protein kinase A binding
Sarcoplasmic/endoplasmic reticulum calcium 5 Q0VCY0 4894 110,532 36 35.1 Bos Ca2+ signaling
ATPase 1
Calcium-transporting ATPase 5 F1MPR3 1890 113,512 22 27.6 Bos Calcium transport
Alpha-1,4 glucan phosphorylase 6 F1MJ28 11,923 97,674 46 54.4 Bos Glycometabolism
Uncharacterized protein(GN ¼ AKAP4) 6 F1MYH5 5115 95,481 5 16.5 Bos Protein kinase A binding
Phosphoglucomutase-1 8 Q08DP0 16,360 61,836 35 55.3 Bos Glycolytic enzyme
Very long-chain specific acyl-CoA 8 P48818 577 71,003 9 28.4 Bos Lipid metabolism
dehydrogenase Oxidoreductase
Pyruvate kinase (Fragment) 8 Q3ZC87 421 62,016 12 34.7 Bos Glycolytic enzyme
Pyruvate kinase (Fragment) 9 Q3ZC87 13,687 62,016 32 54.7 Bos Glycolytic enzyme
Pyruvate kinase (Fragment) 9 B3IVN4 5448 16,688 14 85.2 Bos Glycolytic enzyme
Glucose-6-phosphate isomerase 9 Q3ZBD7 804 63,043 12 25.1 Bos Glycolytic enzyme
Phosphoglucomutase-1 9 Q08DP0 797 61,836 11 33.5 Bos Glycolytic enzyme
Beta-enolase 11 Q3ZC09 34,925 47,409 28 58.5 Bos Glycolytic enzyme
Alpha-enolase 11 F1MB08 18,978 47,596 7 28.1 Bos Glycolytic enzyme
Phosphoglycerate kinase 1 11 Q3T0P6 1242 44,908 13 49.9 Bos Glycolytic enzyme
Creatine kinase M-type 12 Q9XSC6 39,535 43,190 31 80.6 Bos ATP regeneration
Beta-enolase 12 Q3ZC09 19,316 47,409 27 54.6 Bos Glycolytic enzyme
Fructose-bisphosphate aldolase 13 A6QLL8 27,484 39,925 28 73.4 Bos Glycolytic enzyme
Creatine kinase M-type 13 Q9XSC6 22,291 43,190 18 63.3 Bos ATP regeneration
Glyceraldehyde-3-phosphate dehydrogenase 14 P10096 8470 36,073 15 44.1 Bos Glycolytic enzyme
Malate dehydrogenase 14 Q3T145 990 36,700 10 32.6 Bos Glycometabolism
L-lactate dehydrogenase A chain 15 P19858 3556 36,916 10 34.6 Bos Glycolytic enzyme
3 Protein Phosphorylation Affects Meat Color
Adenylate kinase isoenzyme 1 20 P00570 2027 21,764 8 34.5 Bos Creatine energy
metabolism
Myoglobin 23 P02192 5897 17,067 8 57.1 Bos Oxygen transport
a
Protein names and accession numbers were derived from the UniProt database
b
The number of band containing the identified proteins
c
For the proteins identified in more than one band, the highest score was presented
d
Theoretical molecular weight (recorded in UniProt database)
e
Number of matched peptides
f
Percentage of coverage of the entire amino acid sequence
g
Bos, Ovis aries
i
GN, gene name
3.3 Pattern of Sarcoplasmic Protein Phosphorylation in Meat with Different Color. . .
39
protein (GN ¼ AKAP4) and alpha-1, 4 glucan phosphorylase (Table 3.4). The
phosphorylation of proteins in band 5 and band 6 exhibited a negative (P < 0.05)
correlation with L value, b value, the relative content of oxymyoglobin, and the
relative content of metmyoglobin (Table 3.3). Besides, phosphorylation of protein in
band 5 was positively (P < 0.05) correlated with R630/580 value (Table 3.3), while
that of band 6 was negatively (P < 0.05) correlated with a value (Table 3.3). Malate
dehydrogenase was one of the main proteins identified in band 14 (Table 3.4). All
those identified proteins may contribute to differences in meat color, though the
underlying mechanism through which the phosphorylation of those protein influ-
ences meat color stability was still unclear.
3.4 Quantitative Phosphoproteome of Meat

with Different Color
Phosphorylation of some glycolytic enzymes and myoglobin at specific serine

residues may play critical roles in the regulation of meat color stability. A quantita-
tive analysis of protein phosphorylation in ovine LTL muscle with different color
stability was performed using TMT labeling in combination with TiO2
phosphopeptide enrichment. The pH and instrumental color measurement and
phosphoproteomic analysis were carried out. Proteins involved in carbohydrate
metabolism, especially glycolytic enzymes, were the largest cluster of protein
determined to be color-related. The phosphorylation of myoglobin at Ser133 played
a negative role in the regulation of meat color stability. Thus, the phosphorylation of
some glycolytic enzymes and myoglobin at specific serine residues may play critical
roles in the regulation of meat color stability. To further understand the mechanism
by which protein phosphorylation regulated meat color stability, in the present study,
a quantitative MS-based phosphoproteomic analysis of ovine muscle with different
color stability at 24 h postmortem was performed.
3.4.1 Differentially Expressed Phosphoproteins Between

Meat with Different Color
A total of 3412 phosphopeptides were identified, including 1070 phosphoproteins.

Among these, 243 phosphoproteins were significantly different in abundance
between low and high color stability groups, of which 98 proteins were
up-regulated and 145 were down-regulated compared to high color stability group.
One hundred and thirty five phosphoproteins were determined to be different in
abundance between medium and high color stability group, of which 63 proteins
were up-regulated and 72 were down-regulated in medium color stability group. One
hundred and two phosphoproteins were differentially expressed in low and medium
3.4 Quantitative Phosphoproteome of Meat with Different Color 41
color stability muscles, of which 47 proteins were up-regulated and the other 55 were
down-regulated compared to medium color stability group.
In total, 432 unique phosphopeptides were determined to be different in abun-
dance between the three groups of muscle, which were assigned to 273 phosphopro-
teins. To show the phosphorylation pattern of the 432 phosphopeptides, a
hierarchical clustering analysis (HCA) was applied. As shown in Fig. 3.6, the
432 phosphopeptides were marked from red to green in abundance from high to
low. A redder color meant a higher phosphorylation level and a greener color meant
a lower phosphorylation level of the phosphopeptide. Similar color layouts were
observed within the three biological replicates of each group and different color was
C1
C2
C3
A2
A1
A3
B2
B1
B3
0.50
0.33
0.17
0.00
–0.17
–0.33
–0.50
M line
Fig. 3.6 Clustering of differently expressed phosphopeptides in ovine muscles of different color
stability. Note: Muscle samples are displayed in columns and classified by phosphoproteomic
subtypes as indicated by different colors. A1, A2, and A3 are the three samples in high color
stability group. B1, B2, and B3 are the three samples from moderate color stability group. C1, C2,
and C3 represent the three samples in low color stability group. Deeper red represents higher
phosphorylation level and deeper green represents lower phosphorylation level. Protein names are
listed in Supplementary material 1. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.) (Li et al. 2018b)
displayed among different groups, revealing a characteristic pattern in protein

phosphorylation in muscles of different color stability. Specifically, protein phos-
phorylation displayed a “reverse” pattern between the H and L groups. Highly
phosphorylated sites in the H group were lowly phosphorylated in the L group,
vice versa. In order to improve our understanding of the underlying mechanism by
which protein phosphorylation regulated meat color stability, the differentially
expressed phosphoproteins between the H and L groups were selected for bioinfor-
matic analysis in the study. Twenty four putative phosphorylation motifs were
identified by using Motif-X software which included 16 serine motifs, seven thre-
onine motifs, and one tyrosine motif (Supplementary material 2). This indicated that
phosphoproteins with the same phosphorylation motifs might affect meat color
development.
3.4.2 Functional Enrichment Analysis of Differentially

Expressed Phosphoproteins
Functional enrichment analysis of differentially expressed phosphoproteins was

performed to obtain some information about the mechanism regulating meat color
stability (Fig. 3.7). Gene Ontology (GO) terms enrichment revealed that most
differentially expressed phosphoproteins were involved in cellular carbohydrate
metabolic process, glucan metabolic process, polysaccharide metabolic process,
cellular glucan metabolic process, cellular polysaccharide metabolic process,
single-organism carbohydrate metabolic process, carbohydrate metabolic process,
and glyceraldehyde-3-phosphate metabolic process. Kyoto encyclopedia of genes
and genomes (KEGG) pathway enrichment showed that the primary eight pathways
that these phosphoproteins participated were starch and sucrose metabolism, pentose
phosphate pathway, galactose metabolism, glycolysis/gluconeogenesis, fructose and
mannose metabolism, amino sugar and nucleotide sugar metabolism, purine metab-
olism, and biosynthesis of amino acids.
In summary, the functional enrichment analysis revealed that the carbohydrate
metabolism-related proteins were the largest cluster of proteins that were color-
related, showing that meat color stability was probably primarily influenced by
carbohydrate metabolism in postmortem muscle. All differentially expressed phos-
phoproteins of these color-related metabolic processes were selected as key color-
related phosphoproteins. A total of 27 key color-related phosphoproteins were
selected in this study.
3.4 Quantitative Phosphoproteome of Meat with Different Color 43
a
Biosynthesis of amino acids 7
Enriched KEGG Pathways
Purine metabolism 6
p.value
Amino sugar and nucleotide sugar metabolism 4
0.00
Fructose and mannose metabolism 4 0.01
0.02
Glycolysis / Gluconeogenesis 9 0.03
0.04
Galactose metabolism 3 0.05
Pentose phosphate pathway 5
Starch and sucrose metabolism 5
0.0 0.3 0.6 0.9
richFactor
b BP MF CC
0.8 6 6 6 6
8 4
12 12 12 12 p.value
0.6 18 5 5
richFactor
0.015
21
21
0.4 27 26 25 0.010
41
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Enriched GO Terms (Top 20)
Fig. 3.7 The results of GO terms enrichment and KEGG pathway enrichment. (a)The enriched
KEGG pathways of differently expressed phosphoprotein between high and low color stability
group. (b)The enriched GO terms of differently expressed phosphoprotein between high and low
color stability group. (Li et al. 2018b)
3.4.3 Functional Annotation Analysis of Key Color-Related

Phosphoproteins
Gene ontology annotation of the key color-related phosphoproteins was shown in

Table 3.3. The metabolic process grouping of the 27 key color-related phosphopro-
teins was shown in Fig. 3.8. As in Fig. 3.8, 19 out of the 27 key color-related
phosphoproteins were carbohydrate metabolic enzymes, including nine glycolytic
enzymes, six galactose, glycogen, polysaccharide and glucan metabolism enzymes,
Fig. 3.8 Metabolic pathway classification of the 27 key color-related phosphoproteins (Li et al.
2018b)
three phosphorylation regulation related enzymes, and one enzymes of tricarboxylic

acid cycle.
Myoglobin, five purine metabolism and amino acid biosynthesis related enzymes,
and two other proteins were also identified as key color-related phosphoproteins.
More than one phosphopeptides and phosphorylation sites from one key color-
related phosphoprotein were identified in this study. However, not all the
phosphopeptides and phosphorylation sites from one key color-related phosphopro-
tein were differentially expressed in muscles with different color stability.
3.5 Conclusions
Adding additional inhibitors could regulate sarcoplasmic protein phosphorylation

level. The phosphatase inhibition group had a significantly higher phosphorylation
level than control group, and that of kinase inhibition group was significantly lower
than control group. Compared with high phosphorylation level group, the group with
low phosphorylation level had low glycolysis rate, slow pH value decline rate, high
limit pH value, and high redness value. Therefore, protein phosphorylation nega-
tively regulated meat color stability, and it might be caused by regulating glycolysis
metabolism. There was no difference in the whole phosphorylation level of sarco-
plasmic protein from longissimus thoracis et lumborum muscle between the high,
middle, and low meat color-stable groups, and all reached the lowest level at 24 h
after slaughter, and then basically kept the same level. However, there was signif-
icant difference in the phosphorylation level of some sarcoplasmic protein bands
among the three groups. The results of mass spectrometry showed that most of the
meat-color-related protein bands were glycolytic enzymes. Moreover, the Mb phos-
phorylation level was low in the high meat color-stable group, and high in the low
meat color-stable group. There were two possible ways of protein phosphorylation
controlling meat color stability. First, it affected meat color stability by regulating
glycolysis process. Second, Mb phosphorylation might affect meat color stability.
References 45
Three thousand four hundred and twelve phosphorylated peptide segments and 1070
phosphorylated proteins were identified. The results of Gene Ontology and KEGG
Pathway Analysis showed that the phosphorylated differential proteins related to
meat color were mainly involved in the glycometabolism pathway. The key phos-
phorylated proteins that regulated meat color stability included pyruvate kinase,
pyruvate dehydrogenase, glucose phosphomutase, phosphopropyl isomerase, fruc-
tose diphosphate aldolase, enolase, 6-phosphoglucose isomerase, adenylate kinase
isoenzyme 1 and Mb. Most of the phosphorylation of meat color occurred on specific
serine residues. The phosphorylation of these key meat-color-related phosphoryla-
tion proteins at specific sites might be an important factor for phosphorylation to
control meat color stability. In addition, phosphorylation negatively regulated meat
color stability happened on the 133 serine of myoglobin.
Acknowledgments Parts of this chapter are reprinted from Food Chemistry, 240, Li, M., et al.,
Comparative profiling of sarcoplasmic phosphoproteins in ovine muscle with different color
stability, 104-111; Food Chemistry, 249, Li, Z., et al., Quantitative phosphoproteomic analysis
among muscles of different color stability using tandem mass tag labeling, 8-15; Food Chemistry,
219, Li, M., et al., Effects of protein phosphorylation on color stability of ground meat, 304–310.
Copyright (2020), with permission from Elsevier.
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Chapter 4
Protein Phosphorylation Affects Meat
Tenderness
Abstract Tenderness is one of the most important quality attributes especially for
beef and lamb. As protein phosphorylation and dephosphorylation regulate glycol-
ysis, muscle contraction, and turnover of proteins within living cells, it may con-
tribute to the conversion of muscle to meat. The protein phosphorylation level (P/T
ratio) of the tender group increased from 0.5 to 12 h postmortem and then decreased.
The P/T ratio of tough group increased during 24 h postmortem, with higher increase
rate from 0.5 to 4 h postmortem than from 4 to 24 h postmortem. The global
phosphorylation level of tough meat was higher than tender meat at 4, 12, and
24 h postmortem. Protein identification revealed that most of the phosphoproteins
were proteins with sarcomeric function; the others were involved in
glycometabolism, stress response, etc. The phosphorylation levels of myofibrillar
proteins, e.g. myosin light chain 2 and actin, were significantly different among
groups of different tenderness and at different postmortem time points. Protein
phosphorylation may influence meat rigor mortis through contractile machinery
and glycolysis, which in turn affected meat tenderness.
Keywords Ovine muscle · Tenderness · Aging time · Sarcoplasmic proteins ·

Myofibrillar protein · Protein phosphorylation
4.1 Introduction
Tenderness is one of the most important quality indicators that affect consumers’
choices when purchase meat and meat products. Usually, the most unacceptable
thing to consumers is meat with low tenderness (Jeremiah, 1982). Myofibrillar
proteins are the largest protein class in skeletal muscle, and account for 55–60% of
total muscle protein by mass. Myofibrillar proteins are responsible for contractile,
functional, and culinary properties of muscle and meat (Lee, Joo, & Ryu, 2010).
Myofibrillar proteins that regulate muscle contraction could be involved in rigor
mortis development and affect meat tenderness (Hopkins, Toohey, Lamb, Kerr, &
Refshauge, 2011; Huang, Huang, Xu, & Zhou, 2011; Li et al., 2013). Post-
translational modifications are key modulators of protein structure, function,

https://doi.org/10.1007/978-981-15-9441-0_4
50 4 Protein Phosphorylation Affects Meat Tenderness
signaling, and regulation. Protein phosphorylation, a common post-translational

modification, plays a regulatory role in the contraction of skeletal muscle and
myofibrillar protein degradation. Phosphorylated skeletal muscle proteins,
e.g. myomesin and myosin regulatory light chain 2 (MYLC2), were reported to
affect skeletal muscle contraction and protein interaction (Obermann, Gautel,
Weber, & F Rst, 1997; Ryder, Lau, Kamm, & Stull, 2007; Zhi et al., 2005).
Phosphorylation of some myofibrillar proteins has been proved to reduce the deg-
radation of these proteins by calpains (Di Lisa et al., 1995; Zhang, Lawrence, &
Stracher, 1988). Phosphorylation of calpastatin (endogenous calpain inhibitor) ren-
dered it a more effective inhibitor of m-calpain than unphosphorylated calpastatin
(Salamino et al., 1994). However, whether there are differences in protein phos-
phorylation levels and the change in protein phosphorylation levels with postmortem
time between muscles with different tenderness levels is unknown.
Study of the dynamic change of protein phosphorylation postmortem in muscles
with different tenderness levels can identify candidate regulatory proteins and help
to understand the underlying mechanisms of meat quality development. In this
study, ovine muscle samples were divided into two groups according to tenderness.
A combination of sodium dodecyl Diamond-SYPRO Ruby staining and tandem
mass spectrometric strategy was used to detect the phosphoproteins. Phosphoryla-
tion of myofibrillar proteins was comparatively studied between tender and tough
muscles and among muscles at different postmortem time points.
Meat tenderness relies mainly on changes postmortem that affect the contractile
system of the muscle or myofibrils. Warner–Bratzler shear force (WBSF) was one of
the first instrumental methods developed for tenderness measurement, and has
remained the most commonly used one globally (Silva et al., 2015). Another most
extensively studied event taking place in muscles after slaughter is proteolysis,
which leads to myofibrillar degradation (Taylor, Geesink, Thompson, Koohmaraie,
& Goll, 1995). As the main aim of this study was to investigate the myofibrillar
proteins postmortem, the myofibrillar fragmentation index (MFI) was measured as
another indicator to determine tenderness.
The objective of this chapter was to investigate the relation between protein
phosphorylation and meat tenderness. Longissimus dorsi muscles of 40 sheep
(fat-tailed sheep tail sheep) were collected at 0.5, 1, 4, 12, and 24 h postmortem,
and were divided into two groups (high-level-of-tenderness and low-level-of-ten-
derness) based on the shear force measurement and MFI at 24 h postmortem. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was combined
with fluorescence staining in order to analyze the level of phosphorylation of
sarcoplasmic proteins and myofibrillar proteins.
4.2 Changes of Proteins Phosphorylation Levels in Postmortem Muscle with Different. . . 51
4.2 Changes of Proteins Phosphorylation Levels

in Postmortem Muscle with Different Tenderness
There were four samples in the tough group and tender group, respectively. Tender
meat was characterized by significantly lower WBSF (7.57 vs. 13.06 kg, P < 0.01),
higher MFI (99.55 vs. 73.40, P < 0.01), and longer sarcomere length
(1.15 vs. 0.94 μm, P < 0.01) (Fig. 4.1) at 24 h postmortem when compared with
tough meat. Muscle pH values at 4, 12, and 24 h postmortem were also recorded
(Table 4.1).
4.2.1 Changes of Sarcoplasmic Proteins Phosphorylation

Levels in Postmortem Muscle with Different Tenderness
4.2.1.1 SDS-PAGE of Phosphorylated and Total Sarcoplasmic Proteins

of Muscle
The SDS-PAGE electrophoresis patterns of sarcoplasmic proteins in the tough and

tender groups at 0.5 h, 1 h, 4 h, 12 h, and 24 h postmortem were shown in Figs. 4.2
and 4.3. Figure 4.2 was phosphorylated proteins stained with Pro-Q Diamond, and
Fig. 4.3 was SYPRO Ruby-stained sarcoplasmic total proteins. It can be seen from
the images that the bands were straight and clear, and the separation effect was good.
Eighteen clearer bands were selected from the images and their relative optical
densities were analyzed.
Phosphoproteins and the total proteins were detected using Pro-Q Diamond dye
and SYPRO Ruby dye respectively, and the ratio of the fluorescence intensity of
Pro-Q Diamond staining to the fluorescence intensity of SYPRO Ruby staining in
each band was used as the protein phosphorylation level by semi-quantitative
method. Because the protein phosphorylation level (P/T) ratios of the bands 1, 4,
Fig. 4.1 The ultrastructure of myofibril of longissimus muscles in groups of different tenderness
(30000): (a) ultrastructure of myofibril of tender group; (b) Ultrastructure of myofibril of tough
group (Chen et al., 2016)
52
Table 4.1 Meat tenderness indicators (means SD) (Chen, Li, Ni, et al., 2016)
Group WBSF (kg) MFI (24 h) Sarcomere length (μm) pH (4 h) pH (12 h) pH (24 h)
Tender 7.57 7.39b 99.55 4.31a 1.15 0.06a 6.49 0.18 5.91 0.09 5.67 0.06
Tough 13.06 2.42a 73.40 6.20b 0.94 0.08b 6.69 0.22 6.03 0.14 5.71 0.05
Different letters (a, b) within a column indicate that mean values differ (P < 0.01)
4 Protein Phosphorylation Affects Meat Tenderness
Fig. 4.2 SDS-PAGE image displaying the phosphorylated sarcoplasmic proteins present in mut-
ton. Translated from Chen et al. (2015)
6, 7, 8, 10, 15, and 16 were higher than 1.0, which showed the proteins in these
bands had higher phosphorylation levels. The bands 4, 6, and 10 that were inten-
sively stained in the Pro-Q Diamond images were lighter when they were stained in
SYPRO Ruby, while more intensive-stained bands 3, 7, and 9 in the SYPRO Ruby
images were lighter in Pro-Q Diamond staining. These differences indicated that the
Pro-Q Diamond staining was specific for phosphorylated proteins.
4.2.1.2 Comparison of Global Sarcoplasmic Proteins Phosphorylation

It can be seen from Fig. 4.4 that the phosphorylation levels of the tough group and
tender group all increased and then decreased. The highest protein phosphorylation
level in tough group appeared at 4 h postmortem, and the highest protein phosphor-
ylation level in tender group appeared at 12 h postmortem. The global
Fig. 4.3 SDS-PAGE image displaying the total sarcoplasmic protein content present in mutton.
Translated from Chen et al. (2015)
phosphorylation level of tough group was significantly higher than that of tender
group at 0.5 h, 1 h, and 4 h postmortem (P < 0.05). The global phosphorylation level
of sarcoplasmic protein was significantly affected by the tenderness, postmortem
maturity time, and the interaction between tenderness and postmortem maturity
(P < 0.05).
The reason why the phosphorylation level of sarcoplasmic protein in tough group
was higher than that in tender group can be analyzed from the following aspects:
First, according to D’Alessandro and Zolla (2013) and Li et al. (2012), due to their
perspective of “lower phosphorylation-induced enzyme activity,” the higher the
phosphorylation level, the lower the activity of most glycolytic enzymes. The results
showed that the tough group had higher phosphorylation level, and tender group had
lower phosphorylation level. Therefore, under the influence of phosphorylation, the
glycolytic enzyme activity of the tender group was higher, the glycolysis rate was
Fig. 4.4 Comparison of global sarcoplasmic proteins phosphorylation levels between two mutton
groups with different degrees of tenderness. Letters a, b in the same row indicate a significant
difference (P < 0.05). Translated from Chen et al. (2015)
faster, and the pH was lower, which was more conducive to the release and
activation of cathepsins, thus promoting protein degradation, and was beneficial to
meat tenderization (Xia, Li, Chen, Chen, & Zhang, 2014). Second, Doumit and
Bates (2000) pointed out that calpastatin would phosphorylate, and calpastatin
phosphorylation could increase the inhibition of calpain and reduce the degradation
of myofibrillar protein by calpain which was conducive to the tenderization of
muscles. Third, phosphorylation of heat shock protein 27 was a key step in
protecting actin from disruption (Loktionova & Kabakov, 1998). In this experiment,
the phosphorylation level of heat shock protein 27 was higher in tough group. The
more heat shock protein 27 was phosphorylated, the more actin was protected and in
a complete state, which was not conducive to muscle tenderization. Fourth, muscle
contraction increased the phosphorylation level of phosphofructokinase (Luther &
Lee, 1986), and the muscle contraction of tough group was severe, so the phosphor-
ylation of phosphofructokinase was high.
4.2.1.3 Sarcoplasmic Protein Phosphorylation Related to Meat

Tenderness and Postmortem Maturation
There were 18 bands detected and selected for image analysis of the total sarcoplas-
mic protein content (Table 4.2). The phosphorylation levels of bands 3, 6, and
18 were significantly different between different tenderness groups and postmortem
maturity groups (P < 0.05). The phosphorylation levels of bands 1, 2, 5, 9, 14, and
15 were significantly influenced by the interactive effects of tenderness and post-
mortem time (P < 0.05), but the phosphorylation levels of bands 8, 10, and 16 were
56
Table 4.2 Comparison of the sarcoplasmic protein phosphorylation between two mutton groups with different degrees of tenderness. Translated from Chen et al.
(2015)
Tender Tough Effect level (P value)
Tenderness
Band 0.5 h 1h 4h 12 h 24 h 0.5 h 1h 4h 12 h 24 h Tenderness Time Time
1 0.86 0.05 0.86 0.12 1.00 0.07 1.29 0.15 1.02 0.11 0.60 0.06 1.22 0.09 1.38 0.02 0.97 0.04 0.89 0.05 0.951 0.008 0.014
2 0.37 0.02 0.42 0.03 0.39 0.05 0.36 0.04 0.32 0.02 0.54 0.04 1.10 0.07 0.60 0.06 0.28 0.03 0.20 0.01 0.720 <0.0001 0.001
3 0.40 0.03 0.70 0.08 0.51 0.05 0.38 0.03 0.36 0.04 0.46 0.02 0.60 0.05 0.70 0.05 0.39 0.02 0.55 0.03 0.001 <0.0001 0.003
4 0.92 0.08 1.39 0.14 1.45 0.21 1.18 0.04 1.09 0.09 0.74 0.09 0.77 0.12 0.87 0.07 0.84 0.05 0.87 0.04 0.069 0.109 0.234
5 0.40 0.05 0.37 0.02 0.27 0.03 0.19 0.02 0.21 0.03 0.39 0.02 0.35 0.04 0.43 0.03 0.32 0.01 0.26 0.01 0.630 0.001 0.002
6 2.09 0.17 2.40 0.07 3.08 0.24 3.08 0.22 2.19 0.19 1.65 0.08 2.46 0.20 2.46 0.18 2.14 0.13 2.14 0.05 0.011 0.017 0.020
7 0.53 0.06 0.55 0.03 0.58 0.06 0.58 0.05 0.52 0.04 0.36 0.04 0.39 0.02 0.42 0.03 0.39 0.04 0.41 0.03 0.001 0.291 0.235
8 0.56 0.04 0.57 0.06 0.60 0.11 0.77 0.05 0.63 0.02 0.51 0.07 0.66 0.13 0.59 0.04 0.76 0.03 0.62 0.07 0.891 0.010 0.775
9 0.26 0.01 0.36 0.02 0.52 0.006 0.38 0.06 0.27 0.01 0.40 0.05 0.32 0.04 0.29 0.02 0.35 0.04 0.33 0.01 0.562 0.349 0.020
10 0.94 0.07 1.04 0.09 1.06 0.05 1.19 0.15 1.13 0.05 0.91 0.05 1.02 0.05 0.98 0.06 1.16 0.09 1.07 0.07 0.152 0.002 0.687
11 0.38 0.01 0.41 0.03 0.42 0.06 0.42 0.05 0.34 0.01 0.36 0.02 0.40 0.06 0.41 0.05 0.38 0.02 0.38 0.04 0.643 0.096 0.531
12 0.25 0.03 0.26 0.02 0.25 0.04 0.21 0.03 0.12 0.01 0.11 0.02 0.10 0.01 0.13 0.02 0.16 0.03 0.11 0.01 0.799 0.058 0.060
13 0.24 0.02 0.42 0.04 0.50 0.06 0.36 0.03 0.20 0.04 0.16 0.01 0.59 0.07 0.56 0.03 0.34 0.04 0.22 0.01 0.774 0.118 0.937
14 0.43 0.03 0.49 0.05 0.70 0.07 0.58 0.03 0.44 0.06 0.20 0.02 0.29 0.01 0.34 0.04 0.28 0.01 0.58 0.06 0.703 0.002 0.002
15 3.65 0.24 3.39 0.31 3.30 0.05 3.76 0.07 5.33 0.13 2.12 0.11 1.95 0.06 2.24 0.12 2.59 0.17 2.67 0.13 0.471 <0.0001 0.001
16 0.59 0.07 0.57 0.05 0.57 0.04 0.67 0.03 0.77 0.09 0.74 0.04 0.70 0.08 0.72 0.06 0.85 0.07 0.87 0.03 0.725 0.018 0.493
17 0.50 0.06 0.51 0.07 0.76 0.05 0.80 0.09 0.66 0.08 0.17 0.01 0.29 0.04 0.25 0.01 0.42 0.02 0.35 0.02 0.677 0.272 0.754
18 0.24 0.03 0.12 0.01 0.19 0.02 0.20 0.03 0.14 0.02 0.28 0.03 0.21 0.01 0.37 0.05 0.38 0.02 0.33 0.04 0.001 0.002 0.032
Note: Mean SD
only affected by postmortem time (P < 0.05), and the phosphorylation levels of
bands 7 were only affected by tenderness (P < 0.05).
The time of the highest phosphorylation was different in different protein bands.
The phosphorylation level of band 6 and 14 was highest at 4 h postmortem, and the
phosphorylation level of band 8, 10, and 17 was highest at 12 h postmortem. In
addition, the protein phosphorylation level of the band 16 and 18 in the tender group
was higher than that in the tough group, and the protein phosphorylation level of the
band 4, 6, 7, 12, 14, 15, and 17 in the tough group was higher than that in the tender
group. Thus, the global protein phosphorylation level of the tough group was higher,
but not all proteins in the tough group had higher phosphorylation level than the
tender group.
It has been proved that the main protein identified in band 3 (102 ku) might be
glycogen phosphorylase, of which the molecular weight was about 97.4 ku (Huang,
Larsen, Karlsson, Pomponio, & Lametsch, 2011). The molecular weight in this
experiment was large, and the reason might be that the glycogen phosphorylase in
the mutton was highly phosphorylated, causing a change in molecular weight
(Huang, Larsen, Karlsson, Pomponio, & Lametsch, 2011). The same phenomenon
occurred in the study of Huang, Martin, and Lametsch (2012). Glycogen degradation
was carried out by the kinetics process of phosphorylation and dephosphorylation.
Glycogen phosphorylase usually existed in the inactive dephosphorylation state
b. Under the action of phosphorylase kinase and Ca2+, the serine-14 site of glycogen
phosphorylase was phosphorylated, which was converted from inactive b to active a,
and catalyzed the degradation of glycogen to 1-phosphate glucose, and then to
6-phosphate glucose. Band 4 (83 ku) might be phosphofructokinase, which acted
on fructose-6-phosphate and was one of the rate-limiting enzymes for glycolysis.
Therefore, the regulation of this enzyme was a key step affecting glycolysis. On the
one hand, the activation of AMP-dependent protein kinase (AMPK) could cause the
phosphorylation of phosphofructokinase 2, resulting in the activity of phosphofruc-
tokinase 2 and the decreasing production of fructose 2,6-diphosphate, thereby
inhibiting glycolysis and slowing down the drop rate of muscle pH; on the other
hand, phosphorylated phosphofructokinase could alter the affinity for F-actin, which
increased phosphorylation of phosphofructokinase and binding to F-actin (Luther &
Lee, 1986). Band 7 (58 ku) might be pyruvate kinase, which was also one of the rate-
limiting enzymes in the glycolytic pathway. Pyruvate kinase catalyzed the conver-
sion of phosphoenolpyruvate to pyruvate, which can be converted to lactic acid
under the catalysis of lactate dehydrogenase, while nicotinamide adenine dinucleo-
tide (NADH) being oxidized to NAD+. However, studies have shown that AMPK
inhibits the activity of L-pyruvate kinase, thereby inhibiting glycolysis. Band
8 (48 ku) might be β-enolase, the rate-limiting enzyme in the glycolysis process,
which catalyzed the conversion of 2-phosphoglycerate to phospho-enolpyruvate
during glycolysis. It can also catalyze the reverse reaction in the process of glycogen
synthesis, that is, as a phosphopyruvate hydratase, the phospho-enolpyruvate was
converted to 2-phosphoglycerate (Bolten et al., 2008). The band 14 (27 ku) might be
heat shock protein 27, which was increased in expression and phosphorylated to
make it active, and functioned to protect actin and the like. Most of the above
enzymes were related to glycolysis, and most of them in the muscle existed in the
sarcoplasm, and the glycolytic enzymes accounted for two-thirds of the sarcoplasmic
proteins. The rate of glycolysis was determined by the activity of the glycolytic
enzyme. Studies have shown that the increase in glycolytic enzyme activity during
the early postmortem period had an important effect on the muscle maturation
process (Shen, Underwood, Means, Mccormick, & Du, 2007). Whether in vivo or
in vitro, most glycolytic enzymes could be phosphorylated by different kinases, and
phosphorylation affected enzyme activity or stability. By regulating the activity of
enzymes, protein phosphorylation could regulate the postmortem muscle glycolysis
reaction, which in turn affected the postmortem rigor process of the muscle and
played a decisive role of the meat tenderness.
4.2.2 Changes of Myofibrillar Proteins Phosphorylation

4.2.2.1 Comparison of Global Myofibrillar Proteins Phosphorylation
The SDS-PAGE electrophoresis patterns of myofibrillar proteins in the tough and the
tender groups at 0.5, 1, 4, 12, and 24 h postmortem were shown in Fig. 4.5. It could
be seen from the figures that the strips were straight and clear, and the separation
effect was good. Since the protein phosphorylation level (P/T) of band 5, 7, 10, 12,
13, 14, 15, 16, and 21 were higher than 1.0, the proteins in these bands were
considered to be highly phosphorylated. The global protein phosphorylation level
in tender group increased slowly from 0.5 h to 12 h postmortem and then gradually
decreased from 12 h to 24 h. However, the global protein phosphorylation level in
tough group increased rapidly from 0.5 h to 4 h and then increased slowly from 4 h to
24 h (Fig. 4.3). Among them, the phosphorylation level of protein in tough group
was significantly higher than that in the tender group at 4 h, 12 h, and 24 h
postmortem (P < 0.05) (Figs. 4.5 and 4.6).
4.2.2.2 Identification of Phosphorylated Myofibrillar Proteins Related

to Meat Tenderness
There were 21 clearer protein bands excised from gels, then analyzed their relative
optical density and examined them with mass spectrometry. The functional classi-
fications of the proteins were assigned based on the information from the protein
database (UniProtKB), and 34 proteins were finally identified, most of which were
related to sarcomeric and structural function (Table 4.3). There were several sarco-
plasmic proteins in the final experimental results, which may be caused by two
reasons: on the one hand, some sarcoplasmic proteins were contained in the precip-
itated protein pallet during centrifugation due to solubility. On the other hand, some
Fig. 4.5 Gel images of phosphoproteins and total proteins separated by 1-DE. (a) Image of
phosphoproteins stained with Pro-Q Diamond. (b) Image of total proteins stained with SYPRO
Ruby (Chen, Li, Ni, et al., 2016)
Fig. 4.6 Comparison of myofibrillar proteins phosphorylation levels between tender and tough
groups. Letters a, b in the same row at 4, 12, and 24 h indicate a significant difference (P < 0.05)
(Chen, Li, Ni, et al., 2016)
denatured sarcoplasmic proteins were combined with myofibrillar proteins to form

complexes. Some glycolytic enzymes, especially aldolase, glyceraldehyde-3-phos-
phate dehydrogenase, and phosphofructokinase, had significant binding ability with
myofibrillar, such as actin, troponin, and tropomyosin (Arnold, Henning, & Pette,
2005; Clarke & Masters, 1975; Dagher & Hultin, 1975; Melnick & Hultin, 1973).
The main proteins in band 1 were uncharacterized protein (gene name: TTN) and
titin-like protein (TTN). TTN was derived from the M-line and extended along the
myosin fibers, through the A-band of the sarcomere, and finally to the Z-line. TTN
was highly elastic, keeping myosin fibers at the center of the sarcomere during
muscle contraction and relaxation (Ahmed & Lindsey, 2009). Because of the direct
and indirect relationship between TTN and signaling molecules, and multiple phos-
phorylation sites in the Z-line, M-band, and I bands segments, TTN was considered
to be a major regulator of myocyte signaling (Linke, 2008). The proteins in band
2 were uncharacterized protein (gene name: NEB), uncharacterized protein (gene
name: FLNC), and histone H3. The uncharacterized protein (gene name: NEB) was a
protein related to the Z-line function, and the uncharacterized protein (gene name:
FLNC) was an actin-binding protein (UniProtKB in www.uniprot.org). Certain
residues of histone H3, especially the Thr3, Ser10, Thr11, and Ser28 sites, were
highly phosphorylated in the interphase and mitosis of animals, and phosphorylation
of histone H3 was crucial in the regulation of gene expression and chromosome
condensation or isolation (Cerutti & Casas-Mollano, 2009). The proteins in band
3 and 21 were uncharacterized protein (gene name: MYH1), myosin heavy chain
(MHC), myosin light chain 2 (MYLC2). Studies showed that MYLC2
Table 4.3 Unique proteins identified from 1-DE gel and effect analysis (Chen, Li, Ni, et al., 2016)
Band Accession MW Seq. Effect levelh
a c e f
Protein name no.b no. Organism (Da)d Score MP cov.g Function Time Tenderness Time Tenderness
Uncharacterized pro- 1 W5Q754 OS 3,937,785 129,795 4532 63% Sarcomeric < 0.001 0.370 0.003
tein (GNi: TTN) function
Titin-like protein 1 P79395 OS 7620 243 10 61% Sarcomeric < 0.001 0.370 0.003
(fragment) (TTN) function
Uncharacterized pro- 2 W5PFV9 OS 804,157 9886 404 49% Sarcomeric < 0.001 < 0.001 < 0.001
tein (GN: NEB) function
Uncharacterized pro- 2 W5NZK9 OS 278,503 8908 293 66% Sarcomeric < 0.001 < 0.001 < 0.001
tein (fragment) (GN: function
FLNC)
Histone H3 2 B0LRN3 OS 14,760 166 4 43% DNA binding < 0.001 < 0.0001 < 0.001
(fragment)
Uncharacterized pro- 3 W5PPG6 OS 223,600 79,339 2184 75% Sarcomeric 0.018 0.085 0.001
tein (GN: MYH1) function
Myosin heavy chain 3 Q9BE43 OS 11,397 2272 94 55% Sarcomeric 0.018 0.085 0.001
2 (fragment) function
(MYHC2x)
2a (fragment) function
(MYHC2a)
slow (fragment) function
(MYHCs)
Uncharacterized pro- 4 W5PX04 OS 128,132 6791 253 73% Sarcomeric < 0.001 0.437 0.001
tein (GN: MYBPC2) function
Glycogen phosphory- 5 O18751 OS 97,702 1169 42 75% Glycogen < 0.001 < 0.001 < 0.001
lase, muscle form metabolism
4.2 Changes of Proteins Phosphorylation Levels in Postmortem Muscle with Different. . .
(GP)
(continued)
61
62

Band Accession MW Seq. Effect levelh
a c e f
Protein name no.b no. Organism (Da)d Score MP cov.g Function Time Tenderness Time Tenderness
Glycogen phosphory- 5 Q5MIB6 OS 96,881 237 8 14% Glycogen < 0.001 < 0.001 < 0.001
lase, brain form (GP) metabolism
6-Phosphofructoki- 6 W5QDD4 OS 94,291 556 25 36% Glycolysis 0.003 0.025 0.007
nase (PFK)
Heat shock protein α 7 A8DR93 OS 85,077 240 8 14% Stress response < 0.001 0.907 0.117
(HSPα)
Heat shock protein 8 H2DGR2 OS 70,536 225 8 7% Stress response < 0.001 0.008 < 0.001
70 (HSP70) blood
Serum albumin 8 P14639 OS 71,139 2131 79 51% Pressure < 0.001 0.008 < 0.001
(ALB) regulation
Uncharacterized pro- 9 W5PJB6 OS 65,544 2307 97 78% Glucose < 0.001 0.070 0.001
tein (fragment) (GN: metabolism
PGM1)
Glutamate dehydro- 9 Q8MIP8 OS 16,893 124 2 8% Glycogen < 0.001 0.070 0.001
genase (fragment) metabolism
(GDH)
Enolase (fragment) 10 Q9N113 OS 11,952 490 19 79% Glycolysis 0.001 < 0.001 0.001
(ENO)
Beta-actin (fragment) 11 Q9MZW1 OS 25,476 4538 159 85% Sarcomeric 0.019 0.318 0.007
(ACTB) function
Actin (fragment) 11 C7F7J7 OS 38,516 1907 111 48% Sarcomeric 0.019 0.318 0.007
(ACT) function
β-Actin variant 12 D7RIF5 OS 42,052 592 23 17% Sarcomeric 0.024 0.005 0.003
2 (ACTB2) function
Phosphoglycerate 13 B7TJ13 OS 44,936 1461 46 74% Glycolysis < 0.001 0.939 < 0.001
kinase (PGK)
Lactate dehydroge- 13 Q9N109 OS 13,391 871 29 80% Glycolysis < 0.001 0.939 < 0.001
nase A (fragment)
(LDHA)
Aldolase A (frag- 14 Q9N116 OS 15,432 1960 66 67% Glycolysis 0.341 0.521 0.001
ment) (ALDOA)
Glyceraldehyde-3- 15 D7R7V6 OS 36,110 490 17 54% Glycolysis 0.128 0.150 0.306
phosphate dehydro-
genase (GAPDH)
TPM1 16 B2LU28 OS 32,732 1438 45 45% Sarcomeric 0.071 0.654 0.224
function
SLC25A4 17 B2MVW8 OS 33,198 952 44 41% Transmembrane 0.473 0.733 0.353
transport
SLC25A5 17 B2MVW9 OS 33,162 761 29 28% Transmembrane 0.473 0.733 0.353
transport
SLC25A6 17 B2MVX0 OS 33,065 725 28 24% Transmembrane 0.473 0.733 0.353
transport
Adenylate kinase iso- 18 C5IJA8 OS 21,750 331 13 47% Glycogen < 0.001 0.076 0.001
enzyme 1 (AK1) metabolism
Putative tropomyosin 19 Q3BJY7 OS 32,894 43 2 3% Sarcomeric 0.001 0.004 0.002
(TPM) function
Troponin C (TNC) 20 A8WEG2 OS 18,141 1064 27 73% Sarcomeric 0.047 0.150 0.040
function
Fast skeletal myosin 21 B9VGZ8 OS 19,204 2634 78 74% Sarcomeric < 0.001 0.007 0.003
light chain function
2 (MYLC2)
a
Protein names and accession numbers were derived from the UniProt database
b
The number of bands containing the identified proteins
c
OS, Ovis aries
d
Theoretical molecular weight (recorded in UniProt database)
e
For the proteins identified in more than one band, the highest score was presented
f
g
h
Significant levels of different effects on the protein phosphorylation level of individual bands
4.2 Changes of Proteins Phosphorylation Levels in Postmortem Muscle with Different. . .
i
GN, gene name
63
phosphorylated during rigor formation and the ratio of phosphorylated MYLC2 to

unphosphorylated MYLC2 increased at 24 h postmortem, suggesting that MYLC2
phosphorylation might be involved in rigor mortis processes (Huang et al., 2012;
Muroya et al., 2007; Sawdy, Kaiser, St-Pierre, & Wick, 2004). The protein in band
4 was the uncharacterized protein (gene name: MYBPC2).
The phosphorylation of binding protein C disrupted its interaction with myosin
S2, and the distance between myosin S1 and actin was shortened. This shortening of
the distance might increase the possibility of cross-bridge formation, and accelerate
contraction force formation (Taylor et al., 1995). The main proteins in band 5, 9, and
18 were uncharacterized protein (gene name: PGM1), glycogen phosphorylase (GP,
muscle type and brain type), glutamate dehydrogenase (GDH), and adenylate kinase
isoenzyme 1 (AK1) respectively, which were all involved in energy metabolism. For
example, GPb could be phosphorylated by phosphorylase kinase at the serine 14 site,
and its structure was changed and converted to an active state after phosphorylation
(Johnson, 1992; Sprang et al., 1988). The proteins in band 6, 10, 13, 14, and 15 were
6-phosphate fructokinase (PFK), enolase (ENO), phosphoglycerate kinase (PGK),
lactate dehydrogenase A (LDHA), aldolase A (ALDOA), and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), respectively. These enzymes were all glyco-
lytic enzymes, of which PFK was the rate-limiting enzyme of glycolysis, which
could form a complex with actin after phosphorylated. And it was important to
provide energy to cellular components (Cai, Callaci, Luther, & Lee, 1997; Kuo,
Malencik, Liou, & Anderson, 1986; Luther & Lee, 1986). The proteins in band 7 and
8 were heat shock protein alpha (HSPα) and heat shock protein 70 (HSP70). They
were important proteins that protected against the stress conditions, and heat shock
proteins 70 in certain tissues were prone to phosphorylation, such as liver, extensor
digitorum longus, soleus muscles, and myocardium (González & Manso, 2004;
Melling, Thorp, & Noble, 2009). The proteins in band 11 and 12 were β-actin
(ACTB), actin and β-actin variant 2 (ACTB2). Studies have found significant
differences in actin phosphorylation levels in muscles of young rabbits and aging
rabbits, suggesting that aging-induced changes in phosphorylated proteins might
affect muscle contraction (Gannon, Staunton, O’Connell, Doran, & Ohlendieck,
2008). The proteins in band 16 and 19 were the tropomyosin α-1 chain (TPM1)
and putative tropomyosin (TPM). TPM was one of the most phosphorylated proteins
that comprised the thin filaments of muscle. The interactions of adjacent TPM
molecules were more strengthened when phosphorylation occurred, so phosphory-
lation was required for long-range collaborative activation of the thin filaments of
muscle (Rao, Marongelli, & Guilford, 2009). The proteins in band 17 were the solute
carrier family 25 protein (SLC25), including the solute carrier family 25A4 protein
(SLC25A4), the solute carrier family 25 A5 protein (SLC25A5), and the solute
carrier family 25 A6 protein (SLC25A6). They were transporters on the mitochon-
drial membrane and composed of proteins that can transport a variety of molecular
functions (Pebay-Peyroula et al., 2003). The proteins in band 20 were troponin C
(TNC), which can prevent phosphorylase b kinase from catalyzing the phosphory-
lation of troponin I (Cole & Perry, 1975; Perry & Cole, 1974).
According to the functions of identified proteins, these proteins can be divided

into three groups: (1) proteins related to sarcomere function, for example TTN,
MYHC, β-ATBC, TPM1, YNC, MYLC2. (2) Proteins related to glycolysis or
glycogen metabolism, e.g. GP, ENO, PGK, LDHA, ALDOA. (3) Proteins with
other functions, for example, stress-related HSPα and HSP70, and SLC25 proteins
associated with transmembrane transport.
4.2.2.3 Protein Phosphorylation Related to Meat Tenderness

and Postmortem Maturation
The bands in Fig. 4.7 had significant differences in phosphorylation levels which
were affected by tenderness and postmortem time. The P/T ratios of band 5, 7,
10, 12, 13, 14, 15, 16, and 21 were greater than 1, so the proteins in these bands were
highly phosphorylated. The main proteins identified in these bands were GP, ENO,
β-ATBC2, GPK, LDHA, ALDOA, GAPDH, TPM1, and MYLC2, respectively.
The P/T ratios of band 2, 5, 6, 8, 10, 12, 14, 19, and 21 were affected by different
tenderness and postmortem time (P < 0.05, Table 4.3). The phosphorylated proteins
in these bands may have a great impact on the tenderness of the meat. The P/T ratios
of the band 5, 6, 10, 19, and 21 showed similar changing trends between the two
groups, and the ratios of one group was higher than that of the other group (Fig. 4.4).
The protein phosphorylation level of band 2, 8, 12, and 14 initially showed different
patterns of variation between the two groups and eventually tended to be the same
(Fig. 4.7). The P/T ratios of band 1, 3, 4, 11, 13, and 20 were influenced by the
interaction of tenderness and postmortem time (P < 0.05, Table 4.3), while the ratios
of band 7, 9, and 18 were only affected by postmortem time (P < 0.05, Table 4.3).
The trend of protein phosphorylation level over time in the above 18 bands was
shown in Fig. 4.7. The protein phosphorylation level of these bands had significant
differences with postmortem time, but most of these bands had similar changing
trends between the two groups with different tenderness. Thus, postmortem time
should be the main factor affecting the protein phosphorylation level.
The protein phosphorylation levels of band 2, 5, 8, 10, 12, 16, and 21 were
significantly different between the tender and tough groups (P < 0.05). The follow-
ing aspects could explain the relationship between proteins phosphorylation and
muscle tenderness:
First, many studies have demonstrated that MYLC2 phosphorylation was asso-
ciated with muscle contraction. Because myosin light chain could be phosphorylated
under the action of Ca2+/calmodulin-dependent myosin light chain kinase, the
repeated low-frequency stimulation can cause increasing contractility of fast-twitch
muscle (Ryder et al., 2007). The degree of stimulation was positively correlated with
the phosphorylation level of MYLC2 (Klug, Botterman, & Stull, 1982). On the other
hand, MYLC2 phosphorylation was a biochemical memory factor that enhanced
muscle performance (Sweeney, Bowman, & Stull, 1993). Therefore, the higher
phosphorylation level of MYLC2 in the tough group may be one of the most
important reasons for the poor tenderness group of muscles than the other group.
Fig. 4.7 Change patterns of protein phosphorylation level in 18 individual bands (Chen, Li, Ni,
et al., 2016)
Second, protein phosphorylation played an important regulatory role in glycoly-

sis in postmortem muscles. The phosphorylation levels of GP, PFK, ENO, ALDOA,
PGK, and LDHA in the tough group were significantly higher than those in the
tender group (P < 0.05, Table 4.3). The high phosphorylation levels of glycolytic
enzymes caused their decreased enzyme activities (D’Alessandro et al., 2012; Li
et al., 2012). Thus, the glycolytic enzyme activity of the tender group was higher
than that of tough group, which further affected the rate of glycolysis and the
tenderization of muscle (Hopkins, Toohey, Lamb, & Refshauge, 2009).
Third, the P/T ratios of PFK in the tough group were higher than that in the tender
group (Table 4.3). Studies have shown that phosphorylated PFK had a stronger
affinity PFK than the unphosphorylated form. Once a complex was formed, the
enzyme activity was increased as well as the energy production (Luther & Lee,
1986). Stimulation of muscle contraction increased the phosphorylation level of
PFK, which promoted its ability to bind to F-actin (Clarke, Shaw, & Morton, 1980),
which may be the reason that the PFK P/T ratios in the tough group were higher than
the tender group.
Fourth, the interaction between adjacent TPM molecules was enhanced after the
phosphorylation of TPM. Phosphorylation was a requirement for remote collabora-
tive activation of the thin filament (Rao et al., 2009). The TPM phosphorylation level
in the tough group was higher than that in the tender group, so the long-range
cooperative activation of the muscle filaments in tough group was much stronger.
The higher levels of TPM phosphorylation in the tough group may be an important
factor of this group with poorer tenderness than the other group.
4.2.3 Functional Verification of Protein Phosphorylation

Regulation of Myofibrillar Protein
4.2.3.1 Effects of Different Activators on Protein Kinase Activity
After hatching for 0–24 h, the activity of the kinase in the control group decreased,
while the 2-methoxy-1-methylethylacetate (PMA) group and Forskolin group
increased at the beginning and then decreased (Fig. 4.8). After incubation for 1 h,
2 h, and 4 h, the protein kinase activity of PMA group was significantly higher than
the control group (P < 0.05). After 1 h and 4 h, the protein kinase activity of
Forskolin group was significantly higher than control group (P < 0.05). The highest
protein kinase activity of the PMA group and Forskolin group appeared at 1 h,
indicating that protein kinase C activator PMA and Forskolin were involved with
activating kinase activity, leading to the increase of kinase activity. However, the
enzyme activity decreased gradually at 2–24 h, which may be due to the fact that
with the prolongation of postmortem time, the activity of kinase itself decreased
more than the activation of kinase activator, and the activator was consumed
gradually, and the activation ability decreased gradually.
3.0 a
A
Protein kinase activity (U/mg)
2.5 a
bB
2.0 a
A
bA
A
1.5
bB
1.0
0.5
0.0
0h 1h 2h 4h 24 h 0h 1h 2h 4h 24 h 0h 1h 2h 4h 24 h
Control group PMA group Forskolin group
Fig. 4.8 The protein kinases’ activities of different activators’ groups. Letters a, b represent the
significant difference between PMA group and control group (P < 0.05), Letters A, B represent the
significant difference between Forskolin group and control group (P < 0.05). The same as below.
4.2.3.2 Effect of Different Activators on the Phosphorylation Level

of Myofibrillar Protein
The gray value of the electrophoretic graph in Fig. 4.9 was analyzed, and the overall
phosphorylation level of different activator treatment groups was shown in Fig. 4.10.
The whole phosphorylation level of PMA group (protein kinase C (PKC) activated
group) was significantly higher than the control group at 2 h and 4 h (P < 0.05),
Forskolin group (protein kinase A (PKA) activated group) was significantly higher
than control at 2 h and 24 h (P < 0.05). It is suggested that PMA played an important
role after incubation for 2 h and 4 h, while Forskolin played a role at 4 h and 24 h, the
time of action of these two activators was different. The overall phosphorylation
level of the control group slowly increased within 2 h and decreased gradually at
2–24 h, the PMA group increased 0–2 h and then decreased gradually. In Forskolin
group, it increased at 0–1 h and then gradually decreased, but the rate of decline was
lower than the PMA group.
PKA and PKC can promote the phosphorylation level of some proteins
(Fig. 4.11). It was found that PMA promoted the phosphorylation level of band
1 at 2 h, but there was no significant difference between the treatment group and the
control group at other time points (P > 0.05). Forskolin promoted the phosphory-
lation of band 1 at 1 h, 2 h, and 4 h (P < 0.05). PMA promoted phosphorylation of
band 3 at 2 h and 4 h, and of band 5 at 2 h and 24 h, and Forskolin promoted the band
5 at 4 h and 24 h. PMA significantly increased the phosphorylation level of band
7 (P < 0.05). PMA increased the phosphorylation level of band 8 at 2 h, and
Forskolin increased the phosphorylation level of band 8 at 1, 4 h, and 24 h. A new
Fig. 4.9 Gel images of phosphoproteins and total proteins of different activators group separated
by SDS-PAGE. A: Image of phosphoproteins stained with Pro-Q Diamond, B: Image of total
proteins stained with SYPRO Ruby, C: Control group, P: PMA group, F: Forskolin group.
a
1.0
A Control group
A A
a
0.9 bA PMA group
Phosphorylation level (P/T)
a
aB
bB Forskolin group
0.8
0.7
0.6
0.5
0 1 2 4 24
Cultivate time (h)
Fig. 4.10 The global protein phosphorylation level of different activators’ groups. Translated from
Chen, Li, Li, et al. (2016)
protein band appeared at band 6 (25 kDa, Fig. 4.9a) in three different treatment
groups after incubation for 2 h. It was speculated that it may be a small protein
produced by degradation, and Forskolin significantly increased the protein phos-
phorylation level at 4 h and 24 h incubation, and PMA significantly increased the
phosphorylation level of the protein at 24 h (Fig. 4.11).
4.2.3.3 Effect of Different Activators on Protein Degradation
From Fig. 4.9b, it can be observed that a new band appears at the molecular weight
of 25 kDa in the control group at 2 h, as well as the control and PMA group at 24 h.
The MFI of three different treatment groups did not significantly change within
0 h–24 h incubation, and there was no significant difference between the three
treatment groups (Fig. 4.12, P > 0.05).
4.2.3.4 Effects of Different Activators on Muscle Contraction
The results of transmission electron microscopy (TEM) were shown in Fig. 4.13.
The results of the sarcomere length were shown in Table 4.4. With the prolongation
of incubation time, the sarcomere length of each treatment group showed a trend of
slight increase firstly and then sharply decreased, there was no significant difference
in the length of sarcomere between 0–4 h in the control group, and there were
significant differences at 24 h, 2 h, and 4 h (P < 0.05). In the PMA group, there was
no significant difference in the length of sarcomere between 0–4 h, and a significant
difference at 24 h and 2 h (P < 0.05). There was no significant difference in the
Band 1 (Tropomyosin) Control group Band 2 (Myosin heavy chain)

2.0 A
1.4
PMA group 1.2
aA
1.5 aB
a Forskolin group1.0
bB A
aBa
0.8
1.0 0.6
0.5 0.4
0.2
0.0 0.0
0 1 2 4 24 0 1 2 4 24
0.5 0.8
Band 3 (Binding protein 3) Band 4 (Actin)
0.4 a
a 0.6
bA
A
0.3 A
bA
0.4
0.2
0.2
0.1
0.0 0.0
0 1 2 4 24 0 1 2 4 24
0.3 Band 5 (Tropomyosin) A 2.5 Band 6 (25 kD protein)

aA A
a A
a A
2.0
bA aB bB a
0.2 aB a
1.5 bB
1.0
0.1
0.5
0.0 0.0
0 1 2 4 24 0 1 2 4 24
1.0 Band 7 (Troponin T) 2.1 Band 8 (Myosinlight chain 2)

a 1.8 aA a A
0.8 bA
a aB A a
A 1.5 bA A a aB
A aB
bA
0.6 1.2
0.4 0.9
0.6
0.2
0.3
0.0 0.0
0 1 2 4 24 0 1 2 4 24
Cultivate time (h)
Fig. 4.11 The protein phosphorylation level of some important myofibrillar proteins in different
activators’ groups. Translated from Chen, Li, Li, et al. (2016)
length of the sarcomere in Forskolin group within 0–2 h, but a significant difference
between the length of sarcomere at 4 h, 24 h, and 0–2 h (P < 0.05). According to the
comparison of different treatment groups, there was no significant difference in the
length of sarcomere between different treatment groups within 0–2 h of incubation.
At 4 h of incubation, the length of the sarcomere in the activator treatment group was
significantly shorter, and lasted until 24 h, indicating that the activator could result in
the shortening of sarcomere length.
Fig. 4.12 The myofibrillar 60 Control group

fragmentation index of PMA group
different activators’ groups. 50 Forskolin group
Translated from Chen, Li,
40
MFI
Li, et al. (2016)
30
20
10
0
0 1 2 4 24
Cultivate time (h)
4.3 Conclusions
The level of protein phosphorylation was significantly affected by the varying

degrees of tenderness and the different postmortem times. The protein phosphory-
lation level (P/T ratio) of the tender group increased from 0.5 to 12 h postmortem and
then decreased. The P/T ratio of tough group increased during 24 h postmortem,
increasing faster from 0.5 to 4 h postmortem than from 4 to 24 h postmortem. The
global phosphorylation level of tough meat was significantly higher than tender meat
at 4, 12, and 24 h postmortem. The highest level of sarcoplasmic protein phosphor-
ylation in the tough meat and tender meat was observed at 4 and 12 h postmortem,
respectively. Moreover, protein identification revealed that most of the phosphopro-
teins were proteins with sarcomeric function. Protein phosphorylation may influence
meat rigor mortis through contractile machinery and glycolysis, which in turn
affected meat tenderness. PKA and PKC phosphorylated TTN, MYBPC, TPM,
MYLC2 and a protein of 25 kDa. Phosphorylation of some myofibrillar proteins
resulted in reduced degradation of these proteins by the calpains, shortening of
sarcomere length, and muscle contraction.
Agriculture, 96(5), Chen, L., et al., Phosphorylation of myofibrillar proteins in postmortem ovine
muscle with different tenderness, 1474–1483. Copyright (2020), with permission from Elsevier.
Parts of this chapter are translated from Scientia Agricultura Sinica, 49, CHEN, L. J., et al., Protein
phosphorylation on the function of myofibrillar proteins in mutton muscle (Chinese), 1360–1370;
Modern Food Science and Technology, 31, CHEN, L. J., et al., Analyzing the changes in
sarcoplasmic protein phosphorylation with respect to postmortem ageing times in mutton with
different levels of tenderness (Chinese), 95–101. Copyright (2020), with permission from two
journals.
4.3 Conclusions 73
Fig. 4.13 Transmission electron microscopy photos of different activators’ groups in different
time. D0: Control group 0 h; P0: PMA group 0 h; F0: Forskolin group 0 h; D1: Control group 1 h;
P0: PMA group 1 h; F1: Forskolin group 1 h; D2: Control group 2 h; P2: PMA group 2 h; F2:
Forskolin group 2 h; D4: Control group 4 h; P4: PMA group 4 h; F4: Forskolin group 4 h; D24:
Control group 24 h; P24: PMA group 24 h; F24: Forskolin group 24 h. Translated from Chen, Li,
Li, et al. (2016)
Table 4.4 Analysis of the sarcomere length of different activators’ groups. Translated from Chen,
Li, Li, et al. (2016)
Time (h)
Treatment 0 1 2 4 24
Control 1.71 0.06xy 1.62 0.08xy 1.95 0.10x 1.92 0.04a 1.46 0.26aAy
group
PMA 1.71 0.06xy 1.67 0.07xy 1.89 0.29x 1.61 0.10bxy 1.14 0.06by
group
Forskolin 1.71 0.06x 1.75 0.04x 1.74 0.06x 1.56 0.07By 0.96 0.04Bz
group
The letters a, b represent the significant difference between PMA group and control group
(P < 0.05), the letters A, B represent the significant difference between Forskolin group and control
group (P < 0.05), the letters x, y, z represent the significant difference of the same group in different
time points (P < 0.05).
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Chapter 5
Protein Phosphorylation Affects Meat
Water Holding Capacity
Abstract Water holding capacity and its corollary, drip loss, are important meat
quality indicators. The high drip loss muscle had lower pH, higher brightness, higher
shear force value, and shorter sarcomere length than the low drip loss muscle. The
protein phosphorylation level of different drip loss muscles was detected via the
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in this
study. The results showed that the phosphorylation level of sarcoplasmic and
myofibrillar in the high drip loss group was higher than that in the low drip loss
group. Combined with the gray value analysis, the bands with higher phosphoryla-
tion levels of sarcoplasmic proteins were mainly enzymes involved in glycolysis
which could affect the pH; while the bands with higher phosphorylation levels of
myofibrillar proteins were mainly cytoskeletal proteins and contractile proteins. It
can be concluded that the low pH and increased muscle contraction had a major
influence on muscle drip loss.
Keywords Water holding capacity · Protein phosphorylation · Drip loss · Meat

quality · Sarcoplasmic protein · Myofibrillar protein1 · Introduction
5.1 Introduction
Water holding capacity (WHC) and its corollary, drip loss, were important meat
quality indicators. Low drip loss often indicated a high WHC of meat, and the tissue
is juicy, fresh, and tender with a dry surface. The genetics (the halothane and
rendement napole genes), feed composition, pre-slaughter management, and post-
mortem handling could influence the WHC (Apple and Yancey 2013; Bond and
Warner 2007; Huff-Lonergan and Lonergan 2005). At a molecular level, the WHC
was related to the denaturation and oxidation of myofibrillar protein, and actomyosin
interaction (Huynh et al. 2013; Liu et al. 2010). Although studies have found out that
desmin, actomyosin, μ-calpain, integrin, and dystrophin were crucial to the WHC of
meat (Bond and Warner 2007; Huff-Lonergan and Lonergan 2005), it was not
enough to fully reveal the mechanism of drip loss.

https://doi.org/10.1007/978-981-15-9441-0_5
78 5 Protein Phosphorylation Affects Meat Water Holding Capacity
Protein phosphorylation plays an important role in muscles after slaughter.

However, whether there are differences in protein phosphorylation level between
muscles with different WHC levels is unknown.
In this chapter, the longissimus dorsi muscles of three breeds of Boer goat,
Huang-Huai goat, and Boer Laoshan white goat were collected. After measuring
the drip loss, the two samples with the highest and lowest drip loss were selected and
divided into high drip loss group and low drip loss group. The physicochemical
properties and muscle fiber structure of the two mutton groups were compared, and
the protein phosphorylation level was studied by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE).
5.2 Meat Quality Traits with Different Drip Loss Values
5.2.1 Comparison of Physical and Chemical Indicators

Between Different Drip Loss Meat
The pH45 min value, the pH of muscle at 45 min after slaughter, was related to drip
loss (Otto et al. 2004). The pH45 min value of the high drip loss group was
significantly lower than that of the low drip loss group (P < 0.05), but there was
no significant difference among the three breeds within each group (P > 0.05)
(Table 5.1). The L* value of the high drip loss group was significantly higher than
of the low drip loss group for all breeds (P < 0.05), because of more water flowing
onto the muscle surface of high drip loss group and increasing light reflection. There
was no significant difference of a* and b* values between the high drip loss group
and the low drip loss group (P > 0.05).
Delta E (ΔE) was defined as the difference between two colors in L*, a*, and b*
color space. The ΔE values of the high drip loss group were significantly higher than
those in the low drip loss group only for Boer goats (P < 0.05). There was no
difference of Warner Bratzler shear force between the high drip loss group and the
low drip loss group (P > 0.05), perhaps because water loss could lead to a tougher
muscle.
The sarcomere length of the high drip loss group was significantly shorter than
that of the low drip loss group (P < 0.05). With an increase in drip loss, the
sarcomere length decreased, due to greater muscle contraction.
5.2.2 Comparison of Muscle Fiber Ultrastructure Between

Different Drip Loss Meat
In Fig. 5.1a–c were obtained by using a transmission electron microscope to amplify

40,000 to observe the mutton with different drip loss. The differences between the
5.2 Meat Quality Traits with Different Drip Loss Values 79
Table 5.1 Meat quality traits of goat longissimus dorsi muscles with different drip loss values.
(Wang et al. 2016)
High drip loss Low drip loss
Drip loss/% Huang-Huai goat 2.27 0.31a 0.33 0.02b
Boer goat 2.20 0.16a 0.82 0.16b
Boer Laoshan white goat 4.68 0.71a 0.36 0.09b
pH45 min Huang-Huai goat 6.10 0.07b 6.35 0.04a
Boer goat 6.19 0.05b 6.34 0.08a
Boer Laoshan white goat 6.11 0.04b 6.22 0.06a
L* Huang-Huai goat 55.89 3.83a 52.47 2.76b
Boer goat 54.07 1.95a 45.33 0.65b
Boer Laoshan white goat 49.82 1.98a 40.86 1.15b
a* Huang-Huai goat 10.35 0.20 13.29 1.52
Boer goat 10.90 0.35 11.20 0.40
Boer Laoshan white goat 9.47 0.67 12.55 1.41
b* Huang-Huai goat 11.84 0.30 13.35 2.07
Boer goat 11.64 0.35 13.15 0.47
ΔE Huang-Huai goat 54.20 1.29 53.13 1.22
Boer goat 53.01 1.56a 44.04 0.67b
Shear force/kg Huang-Huai goat 7.18 0.60 6.66 1.49
Boer goat 7.47 0.50 7.06 0.67
Sarcomere length/μm Huang-Huai goat 1.27 0.33 1.38 0.31
Boer goat 1.26 0.15 1.36 0.11
Boer Laoshan white goat 1.37 0.26b 1.97 0.30a
Data is reported as mean SD (n ¼ 3). Different superscripts in the same row indicate significant
differences (P < 0.05)
high and low drip loss mutton tissue structure can be visualized. In the low drip loss
group, the sarcomere was relatively complete and clear, the muscle cells were
arranged closely, and there was almost no gap. While the high drip loss group was
partially distorted, deformed, arranged disorderly, z-line partially deviated or bro-
ken, and other proteins near the z-line might be degraded and then attached to the
z-line, then more voids were formed between the muscle cells, and certain metab-
olites were accumulated. In addition, it can be seen from Table 5.1 that the length of
the sarcomere in the high drip loss group was lower than that in the low drip loss
group. As the drip loss increased, the length of the sarcomere decreased, especially
in Boer Laoshan white goats with the highest drip loss. The length of sarcomere
was significantly lower than that in meat samples with low drip loss (P < 0.05),
indicating that the sarcomere became shorter and the muscle contracted in the sample
with high drip loss.
Figure 5.1d–f showed that the arrangement of muscle fibers in mutton with
different drip loss was different. Observed by scanning electron microscopy for
Fig. 5.1 Comparison of muscle fiber ultrastructure between drip loss. (a) TEM of Boer Goats with
low drip loss and high drip loss; (b) TEM of Huang-Huai Goats with low drip loss and high drip
loss; (c) TEM of Boer Laoshan Goats with low drip loss and high drip loss; (d) SEM of Boer
Goats with low drip loss and high drip loss; (e) SEM of Huang-Huai Goats with low drip loss and
high drip loss; (f) SEM of Boer Laoshan goats with low drip loss and high drip loss. Translated
from (He 2014)
500 , it was found that the muscle fibers in the low drip loss group were densely
arranged, and there was almost no gap between the muscle fibers. However, in the
high drip loss group, the muscle fibers were disordered, loose in structure, irregular
in distribution, and the diameter became smaller, the gap between muscle fibers
gradually increased, the homogeneity decreased, and the perimysium also partially
fractured and disintegrated. This indicated that with the increase of drip loss, the
water held in muscle decreased, and the muscle fibers contracted, making the gap
between the muscle fibers become larger, and the integrity of tissue structure was
destroyed.
The connective tissue membrane in the muscle was elastic, which could affect the
maintenance of muscle integrity and damage prevention of muscle fibers. It can be
5.2 Meat Quality Traits with Different Drip Loss Values 81
100μm 100μm
(D)
100μm 100μm
(E)
50μm 50μm
(F)
seen from the figure that the connective tissue of the high drip loss group was
ruptured, indicating that the muscle tissue integrity was lost and the dense structure
was destroyed. The muscle fiber boundary was blurred, the arrangement was disor-
dered, part of the protein was broken, segregated, and disconnected.
5.3 Changes of Proteins Phosphorylation Levels Meat

with Different Drip Loss
5.3.1 Comparison of Sarcoplasmic Proteins Phosphorylation

Levels Between Different Drip Loss Meat
Figures 5.2 and 5.3 showed SDS-PAGE of sarcoplasmic protein in lamb with
different drip loss, the left one was the phosphorylated protein dyed by Pro-Q
Diamond. Figure 5.4 was the global protein dyed by SYPRO Ruby, the bands
were straight and 14 clear bands can be seen in the figure.
As presented in Fig. 5.2 and Table 5.2, the P/T values of the band 3, 5, 6, 7, 8, 9,
10, 11, and 13 were greater than 0.5, which meant that the proteins in these bands
may be phosphorylated to a higher level. The Pro-Q Diamond dye was specific for
phosphorylated proteins. Compared to data in Fig. 5.2, the darker bands in Pro-Q
Diamond dye were lighter in SYPRO Ruby, while the darker bands in SYPRO Ruby
dye were lighter in Pro-Q Diamond.
Compare to the global phosphorylation levels of sarcoplasmic proteins in differ-
ent drip loss samples from the three breeds, Table 5.2 shows that the phosphorylation
level of the high drip loss group was generally higher than that of the low drip loss
group. Among them, the band 7, 10, and 13 in the high drip loss group were
significantly higher than the low drip loss group (P < 0.05), indicating that the
phosphorylation level of individual sarcoplasmic protein in the high drip loss group
was higher.
According to the results of the P/T value of each band in Table 5.2 and other
studies, band 7 (58 kDa) may be pyruvate kinase (PK), which was also one of the
three rate-limiting enzymes in the glycolytic pathway (Huang et al. 2011; Li et al.
2012). PK catalyzed the conversion of phosphoenol pyruvate (PEP) to pyruvate,
Fig. 5.2 SDS-PAGE of phosphorylated protein (left) and total protein in sarcoplasmic protein. 1–2
is low drip loss group, 3–4 is high drip loss group, H is Huang-Huai goat, B is Boer goat, L is
Lushan goat. Translated from (He 2014)
5.3 Changes of Proteins Phosphorylation Levels Meat with Different Drip Loss 83
Fig. 5.3 Phosphorylation of sarcoplasmic proteins between high, low drip loss group in different
varieties. Translated from (He 2014)
Fig. 5.4 SDS-PAGE of total protein in sarcoplasmic protein. Translated from (He 2014)
which can be converted to lactic acid under the catalysis of lactic dehydrogenase
(LDH), and nicotinamide adenine dinucleotide (NADH) was oxidized to NAD+.
Band 10 (40 kDa) was presumed to be creatine kinase (CK), which as an
important phosphokinase, might directly involve in intracellular energy transfer,
muscle contraction, and adenosine triphosphate (ATP) regeneration. With the par-
ticipation of ATP, it can catalyze the phosphorylation of creatine to generate
Table 5.2 P/T value of each Band MW/kDa High drip loss Low drip loss
band in the sarcoplasmic pro-
1 173.571 0.280 0.346
tein. Translated from
(He 2014) 2 107.306 0.428 0.404
3 89.270 0.902 0.895
4 77.150 0.546 0.164
5 68.143 0.817 1.356
6 62.854 0.851 0.862
7 58.769 1.863 1.570
8 46.768 2.283 2.892
9 43.250 0.862 1.744
10 40.473 7.312 0.840
11 37.500 1.214 1.869
12 34.410 0.302 0.620
13 27.250 3.849 1.012
14 24.760 0.526 0.080
phosphocreatine and adenosine diphosphate (ADP). It can also reversibly catalyze

the transfer of high-energy phosphate bond of phosphocreatine to ADP to produce
ATP and creatine. When ATP was rapidly consumed, ADP combined with phos-
phate to form ATP. When ATP was sufficient, creatine combined with phosphoric
acid to form phosphocreatine. Phosphocreatine was a high-energy phosphate sub-
stance that could rapidly recover ATP, a form of energy storage. Studies have shown
that CK and actin, alpha β-crystallin were the substrates of proteolysis during pork
aging. And CK, actin and -crystal protein were the substrates of proteolysis during
pork aging (Lametsch et al. 2002).
Band 11 was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key
enzyme involved in glycolysis, catalyzing the oxidative phosphorylation of glycer-
aldehyde 3-phosphate with the participation of NAD+ and inorganic phosphate. Its
glycolytic activity was regulated by F-actin and plasma membrane. Choudhary et al.
(2000) demonstrated that the GAPDH phosphorylation level increased and the pH
decreased in vivo. GAPDH has also been reported to regulate the structure of the
cytoskeleton by forming actin fiber grids.
Most of the enzymes in the muscle could be found in sarcoplasm, and glycolytic
enzymes accounted for two-thirds of the sarcoplasmic proteins (Scopes and Stoters
1982). Glycolytic enzyme activity was the main factor affecting the decline rate of
muscle pH after slaughter, which further affected the process of postmortem rigor
and played a decisive role in the WHC of meat. The activity of glycolytic enzymes
was regulated by phosphorylation levels. Therefore, studying the phosphorylation
level of postmortem sarcoplasmic proteins was a crucial step in regulating meat
quality.
5.3.2 Comparison of Myofibrillar Proteins Phosphorylation

Levels Between Different Drip Loss Meat
Figures 5.5 and 5.6 showed SDS-PAGE of myofibrillar proteins in lambs with
different drip loss, the left figure was the phosphorylated protein dyed by Pro-Q
Diamond. Figure 5.7 was the global protein dyed by SYPRO Ruby, the bands were
straight and 17 clear bands can be seen in the figure.
In the high drip loss group, the P/T values of the band 2, 3, 5, 7, 9, 10, 11, 15, and
17 were greater than 0.5, while those of band 9, 11, 15, and 17 in low drip loss group
were greater than 0.5 (Fig. 5.5 and Table 5.3). And the P/T values of the band 2, 3,
5, 7, 10, and 17 in the high drip loss group were higher than those in the low drip loss
group, indicating that the phosphorylation level of those bands was higher. The
global phosphorylation level of myofibrillar proteins was consistent with that of
sarcoplasmic proteins, and the phosphorylation level of the high drip loss group was
generally higher than that of the low drip loss group.
There were seven bands in myofibrillar proteins that differ greatly, and five of
them were proteins involved in muscle contraction. Band 2 (140 kDa) should be a
C-protein located in the myofibril A-band, interspersed between thin filaments and
thick filaments. It was a muscle-regulating protein that maintained the stability of
thick filaments (Craig and Offer 1976). Previous study has shown that the phos-
phorylation level of C-proteins was related to the contraction rate of the heart and
regulated the interaction of actomyosin during diastole (Hartzell 1984).
As we can see in Fig. 5.7 and Table 5.3, band 3 (105 kDa) should be α-actin, an
important contractile protein in muscle cells, and one of the major components of the
cytoskeleton. Band 10 (36.7 kDa) may be Troponin T, and band 17 (22 kDa) should
be Troponin I. Troponin was a kind of calcium ion receptor protein, which was
composed of three subunits: I, T, and C. Troponin played an important role in muscle
Fig. 5.5 SDS-PAGE of phosphorylated protein (left) and total protein in myofibrillar protein. 1–2
is low drip loss group, 3–4 is high drip loss group, H is Huang-Huai goat, B is Boer goat, L is
Lushan goat. Translated from (He 2014)
Fig. 5.6 Phosphorylation of myofibrillar protein between high, low drip loss group in different
varieties. Translated from (He 2014)
Fig. 5.7 SDS-PAGE of total protein in myofibrillar protein. Translated from (He 2014)
contraction. When combined with Ca2+, its inhibition of actin and myosin was
weakened, resulting in muscle contraction.
Phosphorylation of the troponin subunit could affect its gliding with myosin,
altered the sensitivity of myofibrils to Ca2+ and the activity of actomyosin ATPase.
When the activation of ATPase by Ca2+ decreased, TnI could re-regulate the binding
of actomyosin (Solaro et al. 1976).
Muscle contraction and relaxation were the results of the mutual sliding of
myofibril thick filaments and thin filaments. A cross-bridge was formed between
the myosin head in the thick myofilament and the actin in the thin filaments. The
Table 5.3 P/T value of each Band MW/kDa High drip loss Low drip loss
band in the myofibrillar pro-
1 182.964 0.647 0.456
tein. Translated from
(He 2014) 2 140.298 0.834 0.423
3 105.555 1.944 0.220
4 85.224 0.107 0.303
5 70.866 2.964 0.257
6 60.343 0.196 0.162
7 55.201 1.330 0.593
8 42.961 0.515 0.330
9 38.417 2.475 5.358
10 36.711 1.418 0.202
11 34.904 1.145 2.250
12 31.394 0.249 0.389
13 29.105 0.114 0.220
14 28.833 0.183 0.204
15 27.112 0.786 0.799
16 24.270 0.350 0.260
17 22.278 0.822 0.661
ATP required for muscle contraction was derived from the hydrolysis and catalysis
of actomyosin ATPase. The activity of the muscle contraction system was regulated
by troponin–tropomyosin located on the thin filament. When the nerve stimulation
signal was transmitted, the troponin combined with the Ca2+ released by the sarco-
plasmic reticulum and changed its configuration, and then drove the thick and thin
muscle filaments to slide and contract, resulting in the combination of actin and
myosin to cause muscle contraction.
According to Fig. 5.7 and Table 5.3, band 5 (70 kDa) may be HSP 70 and band
7 (55 kDa) was presumed to be desmin. HSP 70 was important for muscle function
and played an important role in the regulation of structural proteins. In addition,
HSP70 had the function of anti-apoptosis. Many studies have shown that it was one
of the most important proteins that could affect meat tenderness. Brigitte et al. (2010)
found that its expression decreased in the low shear force group. As a major
component of the intermediate filaments in the muscle cytoskeleton structure,
desmin was closely connected with the z-line, and was a cytoskeletal protein
associated with tenderness (Robson et al. 1981). It interacted with other intermediate
filament proteins to form a reticular formation within the cytoplasm that maintained
the link between the cell’s contractile device and other structural elements. When it
was degraded, the integrity of the myofibrillar protein could be destroyed. However,
if the degradation degree of desmin was low, the intact desmin in the postmortem
muscle could transform the contraction of myofibrillar protein into the contraction of
the whole muscle cell, resulting in water loss (Huff-Lonergan and Lonergan 2005).
5.4 Conclusion
The phosphorylation level of sarcoplasmic and myofibrillar in high drip loss meat
was higher than that in low drip loss meat. The bands with higher phosphorylation
levels of sarcoplasmic proteins were mainly enzymes involved in glycolysis which
had effects on the pH, while the bands with higher phosphorylation levels of
myofibrillar proteins were mainly cytoskeletal proteins and contractile proteins. It
can be concluded that the low pH and increased muscle contraction had a major
influence on drip loss.
Acknowledgments Parts of this chapter are reprinted from Food Science & Biotechnology,
25, Wang, Z., et al., Proteomic analysis of goat longissimus dorsi muscles with different drip loss
values related to meat quality traits, 425-431. Copyright (2020), with permission from Springer.
Parts of this chapter are translated from Master Thesis, 2014, HE, F., P Mechanism of drip loss in
mutton postmortem based on proteomics (Chinese), Yunnan Agricultural University.
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Part II
Mechanism of the Effect of Protein
Phosphorylation on Meat Quality
Chapter 6
Phosphorylation on Postmortem Glycolysis
Abstract Glycolysis, the core metabolic pathway in muscle, is very significant to

the ultimate meat quality. The objective of this chapter was to confirm the involve-
ment of protein phosphorylation on postmortem glycolysis and the possible regula-
tory mechanism from the perspective of enzyme activity. The global
phosphorylation level of sarcoplasmic protein increased early postmortem and
then decreased afterwards in muscle. Protein bands with significantly different
phosphorylation level were identified as glycometabolism related enzymes. By
applying quantitative proteomic tools (isobaric Tags for Relative and Absolute
Quantitation, iTRAQ) and bioinformatics analysis, 24 phosphoproteins clustered
in glycolysis and muscle contraction were identified to be glycolytic rate related
proteins, and phosphorylation of pyruvate kinase at Thr 157 was negatively corre-
lated with glycolytic rate. Furthermore, the animal muscle was treated with a kinase
inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor. Protein phosphorylation
was positively correlated with the activity of glycogen phosphorylase, pyruvate
kinase, and phosphofructokinase. In conclusion, protein phosphorylation played a
role in postmortem glycolysis through the regulation of enzyme activity in muscle.
Protein phosphorylation was related to postmortem glycolysis and glycolytic
enzymes, and the phosphorylation level of glycolytic enzymes may influence its
activities, further regulating postmortem glycolysis.
Keywords Protein phosphorylation · Glycolysis · Glycolytic enzyme · Glycolytic

rate · Phosphoproteomic · Glycogen phosphorylase · Phosphofructokinase · Pyruvate
kinase
6.1 Introduction
The biochemical changes related to postmortem metabolism occurring in muscle

after slaughter were very important to the conversion of muscle into the meat (Paredi
et al. 2012). Generally, they included glycogenolysis converting glycogen into
glucose and glycolysis converting glucose into lactate and H+, with a drop in pH
value. Many literatures have reported that pH decline in the early postmortem was

https://doi.org/10.1007/978-981-15-9441-0_6
94 6 Mechanism of the Effect of Protein Phosphorylation on Postmortem Glycolysis
critical to meat quality development (Huang et al. 2011; Lindahl et al. 2006). Rapid
and excessive glycolysis leaded to pale, soft, and exudative (PSE) meat, while
insufficient glycolysis resulted in dark, firm, and dry (DFD) meat (Shen et al.
2006a; Immonen and Puolanne 2000). Very minute differences in the ultimate pH
and rates of pH decline could cause great differences in meat quality. The underlying
mechanisms are still not fully understood, however, pH and carbohydrate metabo-
lism were always of key importance (Pösö and Puolanne 2005).
Posttranslational modifications had the ability to regulate protein structure, func-
tion, signalling, and activity. Protein phosphorylation, one of the most frequent
posttranslational modification, was a key regulator in glycolysis metabolism (Graves
and Krebs 1999). Most glycolytic enzymes were phosphoproteins, including the
three rate-limiting enzymes, hexokinase, phosphofructokinase (PFK), and pyruvate
kinase. PFK was activated after phosphorylation by AMP-activated protein kinase
(AMPK) to upregulate glycolysis and maintain intracellular ATP homeostasis in the
ischemic heart (Marsin et al. 2000). Phosphorylation of phosphoglycerate mutase at
threonine 466 significantly increased the enzyme activity (Gururaj et al. 2004).
Consistently, glyceraldehyde-3-phosphate dehydrogenase purified from the skeletal
muscle of hibernating mammals showed lower phosphorylation levels compared to
the control (Bell and Storey 2014). However, these studies have not been carried out
in postmortem muscle, as differences may exist in the machinery regulating glycol-
ysis antemortem and postmortem. During the postmortem time under anaerobic
conditions, Shen et al. (2006b) also found out that AMPK regulated glycolysis in
postmortem muscle at least partially through phosphorylation. Lametsch et al.
(2011) proved that the pH decline of the rendement napole (RN) genotype could
be a consequence of phosphorylation of glycolytic enzymes during the postmortem
metabolism. All these studies showed that protein phosphorylation had a wide-range
effect on the postmortem conversion of muscle to meat. Thus, it is necessary to have
an overview of protein phosphorylation in postmortem muscle to understand its
function.
The influence of protein phosphorylation on meat quality, including meat ten-
derness, color stability, juiciness, and so on, has been intensively studied (Li et al.
2017; Chen et al. 2016). The influence of phosphorylation on glycolysis has been
proposed to be one possible mechanism. To further understand the mechanism
regulating postmortem changes and meat quality development, especially the bio-
chemistry of postmortem glycolysis, we profiled phosphoproteins in ovine muscle
with the different glycolytic rate in the early postmortem to see the role of protein
phosphorylation on the glycolytic rate and the effect of related glycolytic enzymes in
ovine muscle. Furthermore, we profiled protein phosphorylation in postmortem
muscle with kinase/phosphatase inhibitors to verify the effect of protein phosphor-
ylation on glycolysis and elucidate the regulatory mechanisms from the perspective
of enzyme activity. This research broadened our knowledge about the regulation of
protein phosphorylation in postmortem muscle.
6.2 The Effect of Sarcoplasmic Protein Phosphorylation on Glycolysis in Postmortem. . . 95
6.2 The Effect of Sarcoplasmic Protein Phosphorylation

on Glycolysis in Postmortem Muscle
The phosphorylation level of sarcoplasmic proteins was investigated in relationship

to the glycolysis rate in postmortem ovine muscle. The longissimus thoracis muscles
of 60 sheep were removed immediately after evisceration and kept at 4 C. The pH
values of muscle were determined at 0.5, 2, 6, 12, 24, 48, and 72 h postmortem
(PM) with a portable pH meter (Testo205 pH meter, Lenzkirch, Germany). Muscle
samples at 0.5, 2, 6, 12, 24, 48, and 72 h PM were collected from the right side of
carcasses and stored at 80 C. Muscle samples were divided into three groups: fast
pH decline (F) group (pH6h < 5.75), moderate pH decline (M) group
(5.75 < pH6h < 6.20) and slow pH decline (S) group (pH6h > 6.20) based on muscle
pH values at 6 h (pH6h) postmortem. Muscle pH values at 0.5 and 6 h PM were
significantly different among the three groups (P < 0.01).
6.2.1 Global Phosphorylation Level of Sarcoplasmic Protein

with Different Glycolytic Rates
Sarcoplasmic proteins were stained with Pro-Q Diamond and SYPRO Ruby after
separating by SDS-PAGE to quantify protein phosphorylation by densitometric
analysis (Fig. 6.1). Individual protein bands differences in phosphorylation levels
were observed among the three groups. As shown in Fig. 6.2, the global phosphor-
ylation level of sarcoplasmic proteins in all the three groups of muscles increased
early postmortem and then decreased afterwards. The highest phosphorylation level
in the F group was observed at 2 h PM, which was not determined in the M and the S
groups until 6 h PM. The global phosphorylation level of sarcoplasmic proteins was
significantly lower in the F group than in the S and the M groups at 6 h PM
(P < 0.05).
6.2.2 Phosphorylation Analysis of Individual Protein Bands
Totally 20 protein bands were detected via significance analysis. The phosphoryla-
tion levels of 8 protein bands were significantly different among the three groups
(Fig. 6.3). The phosphorylation levels of band 4 and 8 were significantly different at
0.5 h PM, whereas band 8, 11, 13, and 18 were significantly different at 2 h PM
among the three groups. In general, the phosphorylation level of band 4, 11, 13, and
18 was higher in the F group, while band 8 was higher in the S group.
1
2
3
4*
5
6*
7
8*
9
10*
11*
12
13*
14
15
16
17
18*
19*
20 (A)
1
2
3
4* 5
6* 7
8*
9
10*
11*
12
13*
14
15
16
17
18*
19*
20 (B)
Fig. 6.1 Gel-base analysis of sarcoplasmic protein phosphorylation. (a) Images of gels stained with
Pro-Q Diamond. (b) Images of gels stained with SYPRO Ruby. Bands marked with * were selected
for protein identification by LC-MS/MS (Chen et al. 2018)
Correlation analysis between glycolytic rate attributes and phosphorylation level

was conducted and the results were shown in Table 6.1. Band 6, 8, 11, 13, and
19 were found to correlate with the glycolytic rate (pH, glycogen, lactate acid). The
glycolytic rate was negatively correlated with band 6, 11, 13, and 19, and positively
correlated with band 8.
Fig. 6.2 Quantification of global phosphorylation levels of sarcoplasmic proteins. Different letters
(x, y) at the same time points are significantly different between groups (P < 0.05). Different letters
(A–C) are significantly different at different PM time (P < 0.05) (Chen et al. 2018)
6.2.3 Sarcoplasmic Protein Identification with Different

Phosphorylation Level of the Three Glycolytic Rate
Groups
The eight protein bands significantly different in phosphorylation levels were

excised and identified by LC-MS/MS. Protein peptides were matched with Ovis
aries database, and the proteins with a very high percentage of sequence coverage or
a very high score were selected. In total, 17 unique proteins, molecular weight
ranging from 21.75 to 118.18 kDa, were identified (Table 6.2). The name of all
identified proteins was confirmed using Protein Knowledgebase (UniProtKB in
www.uniprot.org). Most of the sarcoplasmic proteins identified were clustered in
glycometabolism. Glycogen phosphorylase, creatine kinase, 6-PFK, enolase 3, phos-
phoglucomutase 1, glucose-6-phosphate isomerase, pyruvate kinase, phosphoglyc-
erate kinase, triosephosphate isomerase, and AKI 1 were confirmed in the eight
bands with significantly different phosphorylation level, which might be associated
with the glycolytic rate.
There were significant correlations between the glycolytic rate and the phosphor-
ylation levels of band 6, 8, 11, 13, and 19, where band 8 was positively correlated,
and the others had a negative correlation.
PFK, identified in band 6, was the first committing step in the glycolytic pathway.
The phosphorylation of PFK altered the kinetic behavior of the enzyme and regu-
lated the compartmentalization of the enzyme which was observed in vitro and vivo
studies (Luther and Lee 1986). Some studies have demonstrated that PFK was
activated after phosphorylation by AMPK, contributing to maintain high glycolysis
(Marsin et al. 2000).
Fig. 6.3 The phosphorylation levels of 8 glycolytic rate related protein bands. The black, white,
and dashed bars represent the fast glycolytic rate group, moderate glycolytic rate group, and slow
Table 6.1 Pearson correlation coefficients between glycolytic rate attributes and the phosphory-
lation level of total sarcoplasmic proteins or individual protein bands (Chen et al. 2018)
pH value Glycogen content Lactate acid
Global phosphorylation level 0.17039 0.17906 0.05086
Band 1 0.46893* 0.42422 0.56162*
Band 2 0.23692 0.32326 0.25137
Band 3 0.20943 0.18129 0.28011
Band 4 0.03318 0.07177 0.38464
Band 5 0.28724 0.13035 0.35938
Band 6 0.80322** 0.83210** 0.70452**
Band 7 0.09155 0.13035 0.10880
Band 8 0.52808* 0.49561* 0.58264*
Band 9 0.24468 0.33500 0.30962
Band 10 0.01289 0.06243 0.23078
Band 11 0.93183** 0.91226** 0.69681**
Band 12 0.13858 0.08518 0.14148
Band 13 0.82957** 0.85672** 0.94551**
Band 14 0.38669 0.38515 0.40643
Band 15 0.23815 0.27530 0.21473
Band 16 0.49102* 0.37941 0.23656
Band 17 0.18796 0.33037 0.24943
Band 18 0.06107 0.01097 0.28540
Band 19 0.64045** 0.53918* 0.31309
Band 20 0.19653 0.21542 0.37442
Note: Significant levels: *, 0.01 < p < 0.05; **, P < 0.01
Enolase, identified in band 11 and 13, was a key enzyme in the glycolysis
pathway. Enzyme activity assay demonstrated that the hyper-phosphorylation of
a-enolase reduced its activity (Jin et al. 2008). The studies on phosphorylated
enolase showed that the forward reaction was activated and the backward reaction
was inhibited (Nettelblad and Engström 1987).
AKI, identified in band 19, was a key enzyme that could maintain muscle energy
homeostasis. As the phosphotransferase, several isoforms of Adenylate kinase
existed in mammalian tissues were well documented (Dzeja and Terzic 2009).
Marjan Amiri reported that AKI catalyzed the phosphorylation of AMP and CMP
with GTP as the phosphate donor, while AMP, dAMP, CMP, and dCMP were all
phosphorylated when ATP was the phosphate donor (Amiri et al. 2013). Three
phosphorylation sites, including S38, S178, and S181, have been identified in
adenylate kinase in human skeletal muscle (Højlund et al. 2009). Therefore, AKI
⁄
Fig. 6.3 (continued) glycolytic rate group, respectively. Data are presented as mean standard
deviation. Different letters (x, y) at the same time points are significantly different between groups
(p < 0.05). (Chen et al. 2018)
Table 6.2 Identified proteins in the 8 glycolytic rate related protein bands by LC-MS/MS (Chen et al. 2018)
100
Accession Sequence
No. noa Protein name Scoreb Massc Matchesd coveragee Uniprotf
Band W5NVR5 Uncharacterized protein 9308 118,179 555(345) 48 Calcium-transporting ATPase
4
O18751 Glycogen phosphorylase 9284 97,702 524(334) 80 Glycogen phosphorylase, muscle form
W5Q3Y4 Calcium-transporting 4030 107,971 260(177) 43 Calcium-transporting ATPase
ATPase
A8DR93 Heat shock protein 4742 85,077 249(166) 56 Submitted name: Heat shock protein 90 alpha fam-
ily class A member 1
Band W5QAA9 Uncharacterized protein 11,188 85,981 604 (444) 56 Aconitate hydratase, mitochondrial
6 (fragment)
O18751 Glycogen phosphorylase 6324 97,702 365 (262) 67 Glycogen phosphorylase, muscle form
W5QDD4 6-phosphofructokinase 5696 95,200 311 (238) 52 ATP-dependent 6-phosphofructokinase
Band W5PJB6 Uncharacterized protein 38,705 65,544 1730 (1397) 87 Submitted name: Phosphoglucomutase 1
8 (fragment)
W5QC41 Pyruvate kinase (fragment) 6407 62,180 297 (220) 65 Pyruvate kinase
W5P323 Glucose-6-phosphate 4885 63,079 227 (164) 71 Glucose-6-phosphate isomerase
isomerase
Band W5P323 Glucose-6-phosphate 18,487 63,079 975 (695) 90 Glucose-6-phosphate isomerase
10 isomerase
P51977 Retinal dehydrogenase 1 5724 55,417 275 (206) 66 Retinal dehydrogenase 1
W5QC41 Pyruvate kinase (fragment) 3667 62,180 210 (136) 67 Pyruvate kinase
Band W5P663 Uncharacterized protein 10,328 47,382 502 (384) 73 Submitted name: Enolase 3
11
W5PIG7 Uncharacterized protein 8853 48,565 415 (307) 69 Submitted name: Uncharacterized protein
W5PIG6 Uncharacterized protein 8823 49,727 405 (305) 69 Submitted name: Uncharacterized protein
W5PJ69 Uncharacterized protein 8209 43,213 469 (324) 82 Submitted name: Creatine kinase, M-type
B7TJ13 Phosphoglycerate kinase 7134 44,936 436 (313) 87 Phosphoglycerate kinase
6 Mechanism of the Effect of Protein Phosphorylation on Postmortem Glycolysis
W5PN24 Elongation factor 1-alpha 7057 56,244 413 (289) 58 Submitted name: Uncharacterized protein
W5PEP7 ATP synthase subunit beta 6340 56,174 261 (202) 70 ATP synthase subunit beta
W5PD15 Elongation factor 1-alpha 4149 50,451 267 (186) 53 Elongation factor 1-alpha
Band B7TJ13 Phosphoglycerate kinase 23,359 44,936 1256 (926) 92 Phosphoglycerate kinase
13
W5NYJ1 Uncharacterized protein 14,982 42,366 716 (558) 90 Submitted name: Uncharacterized protein
W5PG09 Phosphoglycerate kinase 13,225 45,071 620 (478) 37 Phosphoglycerate kinase
W5PJ69 Uncharacterized protein 11,124 43,213 641 (425) 88 Submitted name: Creatine kinase, M-type
W5PZK7 Uncharacterized protein 10,977 42,381 617 (479) 84 Submitted name: Uncharacterized protein
W5P663 Uncharacterized protein 7971 47,382 391 (289) 62 Submitted name: Enolase 3
D7RIF5 Beta-actin variant 2 6885 42,052 450 (347) 64 Submitted name: Beta-actin variant 2
W5Q0P3 Uncharacterized protein 5943 45,905 371 (265) 74 Submitted name: Creatine kinase, mitochondrial 2
Band W5P5W9 Triosephosphate isomerase 13,232 23,021 575 (413) 91 Triosephosphate isomerase
18 (fragment)
Band C5IJA8 Adenylate kinase isoen- 8047 21,750 671 (425) 84 Adenylate kinase isoenzyme 1
19 zyme 1
a
Accession numbers were derived from the UniProt database.
b
For the proteins identified in more than one band, the highest score was presented.
c
Theoretical molecular weight (recorded in UniProt database).
d
Number of matched peptides.
e
Percentage of coverage of the entire amino acid sequence.
f
The result searching on the website, http://www.uniprot.org/.
6.2 The Effect of Sarcoplasmic Protein Phosphorylation on Glycolysis in Postmortem. . .
101
was critical in glycolysis not only for the phosphorylation itself, but also for
catalyzing the phosphorylation of the other phosphates.
Therefore, protein phosphorylation may be one of the reasons for the difference in
glycolysis rate at 0.5 h PM. Further changes of phosphorylation level at different PM
time, especially the regulation of kinase and phosphatase, will provide more clues to
explore its precise mechanism.
6.3 Quantitative Phosphoproteomic Analysis of Muscle

with Different Postmortem Glycolytic Rate
Protein phosphorylation of ovine muscle at 0.5 h postmortem was profiled in

relationship to glycolytic rate. Quantitative proteomic tools isobaric Tags for Rela-
tive and Absolute Quantitation (iTRAQ) and bioinformatics analysis were used to
further understand the mechanisms regulating postmortem changes.
6.3.1 Phosphoprotein Identification and Motif Analysis
A total of 1905 phosphopeptides were identified, which were assigned to 704 phos-
phoproteins. As to significant difference, 97 phosphopeptides were determined to be
different in abundance between F and M groups, of which 67 peptides were
downregulated and 30 were upregulated in F group compared to the M group.
Forty one phosphopeptides had a significant difference in abundance between M
and S groups, of which 15 peptides were downregulated and 26 were upregulated
compared to S group. Eighty nine phosphopeptides were differently expressed in F
and S groups, of which 51 peptides were downregulated and 38 were upregulated
compared to S group. A total of 116 unique phosphopeptides, matching to 98 phos-
phoproteins and containing 188 phosphorylation sites, were significantly different in
abundance among the three groups after one-way ANOVA analysis. Generally,
160 phosphoserine, 26 phosphothreonine and 2 phosphotyrosine residues (ratios of
85.11%, 13.83% and 1.06% respectively) were identified. Sixteen putative phos-
phorylation motifs were identified after analyzing with the Motif-X software, 13 ser-
ine motifs PS and 3 threonine motifs PT were included (Fig. 6.4). It is logical to
deduce that proteins phosphorylated at these sites may play a role in the glycolysis
pathway.
....P.SP..... .....SD.E...
. .....
...R..SP ......SP.....
0
0
0
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
......SE.E... ......S..D... ...R..S...... ......S..E...
0
0
0
0
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
......S..S... ...S..S...... ......S.S.... ......S.E....

0
0
0
0
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
......SS..... ......TP..... ......TE..... ......T.S

....
0
0
0
0
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
-6
-5
-4
-3
-2
-1
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
+1
+2
+3
+4
+5
+6
6.3 Quantitative Phosphoproteomic Analysis of Muscle with Different Postmortem. . .
Fig. 6.4 Motif analysis among the three glycolytic rate groups. Note: The Logo-like representations of putative motifs are identified from all unambiguous
phosphorylation sites. The height of the residues represents the frequency with which they occur at the respective positions. The color of the residues represents
their physicochemical properties
103
6.3.2 Hierarchical Clustering Analysis
The 116 phosphopeptides with a significant difference in phosphorylation level were

applied to hierarchical clustering analysis in Fig. 6.5. All 116 phosphopeptides were
shown in 116 rows. Different colors in the same row represented phosphorylation
levels of one phosphopeptide among the three groups. Red color represented a high
abundance and a high phosphorylation level, while the green color meant a low
abundance and a low phosphorylation level. The phosphorylation level of all the
116 phosphopeptides were significantly different among the three groups, especially
between the F and S groups. The phosphopeptides with high phosphorylation levels
in the F group (upper part in Fig. 6.5) had a low phosphorylation level in the S group,
F1 F2 F3 M1 M2 M3 S1 S2 S3
Fig. 6.5 Hierarchical clustering analysis among three glycolytic rate groups. Note: Muscle samples
are displayed in columns and classified by phosphoproteomic subtypes as indicated by different
glycolytic rates. Two random samples in the same group were mixed as a replication. F1, F2, and F3
are the three replications in the fast glycolytic rate group. M1, M2, and M3 are the three replications
in the moderate glycolytic rate group. S1, S2, and S3 are the three replications in the slow glycolytic
rate group. The same row represents one phosphopeptide, and different color represent phosphor-
ylation levels. A redder color means a higher phosphorylation level and a greener color means a
lower phosphorylation level of the phosphopeptide (Chen et al. 2019b)
6.3 Quantitative Phosphoproteomic Analysis of Muscle with Different Postmortem. . . 105
while the phosphopeptides with low phosphorylation level in the F group (lower part
of Fig. 6.5) had a high phosphorylation level in the S group. At the bottom, a few
phosphopeptides were expressed irregularly among the three groups. Hierarchical
cluster analysis visualized the specificity and reproducibility of the experiment.
6.3.3 Functional Enrichment Analysis
Gene Ontology (GO) terms enrichment and Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway enrichment were conducted to obtain the important
information about the regulation mechanism of glycolysis (Fig. 6.6). All the differ-
entially expressed phosphoproteins were used to perform protein–protein interaction
network analysis and three different clusters were showed in Fig. 6.7. The largest
one was the muscle contraction related proteins, and the closest interaction one was
the glycolytic enzymes. Pyruvate kinase, phosphoglucomutase 1, enolase2, eno-
lase3, and fructose-bisphosphate aldolase were identified to be the glycolytic rate
related phosphoproteins in the present study, which were detected to be phosphor-
ylated at Thr157, Ser402, Ser177, Ser176, S124, and Ser127, respectively, whose
phosphorylation level was significantly different among the three groups.
6.3.4 Quantitative Analysis of Phosphopeptides
More than one phosphopeptides and phosphosites were identified in the 24 glycolytic
rate related phosphoproteins in the present study. Totally, 390 phosphopeptides were
identified from 24 phosphoproteins, in which 32 phosphopeptides were different in
phosphorylation levels among the three groups (Table 6.3). Moreover, the
phosphopeptides and phosphosites revealed different phosphorylation levels
among the three groups.
Glycolysis is a sequence of enzymatic reactions that are determined by the
activities of glycolytic enzymes. Several studies revealed that the glycolytic enzymes
influenced the transition process of muscle to meat for its activities changed after
slaughter (Werner et al. 2010; Huang et al. 2011). Protein phosphorylation played a
regulatory role in protein structure, function, signaling, and activity regulation. Most
glycolytic enzymes have been reported to be phosphorylated and phosphorylation
increase enzymes’ activity or stability (Sale et al. 1987; Reiss et al. 1986). Pyruvate
kinase, phosphoglucomutase 1, enolase2, enolase3, and fructose-bisphosphate aldol-
ase were identified to be the glycolytic rate related phosphoproteins in the present
study, which were detected to be phosphorylated at Thr157, Ser402, Ser177, Ser176,
S124, and Ser127, respectively. Their phosphorylation level was significantly dif-
ferent among the three groups, with a significantly higher level in M and S groups
than in the F group (Table 6.4).
30
0.25
0.21
p.value
20 0.04
0.03
0.32
0.02
10 0.01
0.38
0.45 0.42 0.42 0.42 0.42 0.42 0.33
0.67 0.5 0.4
0.6 1 1 1 0.75
1
(A)
(B)
Fig. 6.6 GO terms enrichment and KEGG pathway enrichment of differently phosphorylated
proteins in muscles of different glycolytic rate groups. (a) The enriched GO terms of differently
expressed phosphoprotein among the three groups. (b) The enriched KEGG pathways of differently
expressed phosphoprotein among the three groups. Note: The abscissa in the a indicates the
enriched GO function, BP, MF, and CC represent the biological process, molecular function, and
cellular component, respectively. The ordinate in the b indicates the significant KEGG pathway.
The numbers above the bars called rich factor, it indicates the ratio of proteins corresponding to the
phosphopeptides significantly different in phosphorylation level to all identified proteins (Chen
et al. 2019b)
Pyruvate kinase, a rate-limited glycolytic enzyme, catalyzed the conversion of

phosphoenolpyruvate to pyruvate irreversibly. Phosphorylation at Thr157 showed a
significant difference among the three groups, with phosphorylation level being
6.3 Quantitative Phosphoproteomic Analysis of Muscle with Different Postmortem. . . 107
ENO1 PGM1
PDLIM3 ENO2
PDE4B
ALDOC
ENO3
HSP90AA
PKM
HRC
LMOD2
MYH2
ATRX
LDB3 TPM1
TPM2
NEB MYBPC
MYOT
1
MYL2
TTN
PLN
MYLK
2
FHL1
Fig. 6.7 Protein–protein interaction networks of identified glycolytic rate related phosphoproteins
in ovine muscle. Different clusters of interacting proteins were identified using STRING to obtain a
high confidence evidence network (Chen et al. 2019b)
higher in the S group and lower in the F group. The trend was consistent with Chen’s
result (Chen et al. 2018). It has been reported that pyruvate kinase had two isoforms
in normal muscle. Isoforms 2 arose from isoform 1 through phosphorylation.
Phosphorylation of pyruvate kinase could result in an additional, more acid-stable
enzyme isoform, and maintain high activity in PSE meat (Zhang and Liu 2017).
Therefore, the low phosphorylation level in the F group at early postmortem
indicated the high activity of pyruvate kinase.
The activity of phosphoglucomutase 1 may alter after phosphorylation, which can
stimulate the drop of pH value (Gururaj et al. 2004). Fructose 1,6-bisphosphate
aldolase was one of the phosphoproteins that may be directly linked to postmortem
pH decline (D’alessandro and Zolla 2013). The activity of enolase 1 was reduced at
hyper-phosphorylation condition (Jin et al. 2008). Based on these reports, we
supposed that the phosphorylation level of these glycolytic enzymes was related to
the glycolytic rate at early postmortem.
Table 6.3 The 32 phosphopeptides and their phosphosites of 24 glycolytic rate related phospho-
proteins (Chen et al. 2019b)
Sequence (significantly Location in
No Accessions Phosphoproteins Phosphosite in peptide F M S
different among groups) phosphoproteins
1 W5NQP9 Fructose-bisphosphate aldolase gILAADEsVGsMAk S(8): 100.0; S(11): 100.0 S124,S127
2 W5QC41 Pyruvate kinase gPEIRtGLIk T(6): 100.0 T157
3 W5PJB6 Phosphoglucomutase 1 fFGNLMDAsk S(9): 100.0 S402
4 W5P5C0 Enolase 2 lAMQEFmILPVGAEsFR S(15): 100.0 S177
5 W5P663 Enolase 3 lAMQEFmILPVGAsSFR S(14): 97.2; S(15): 2.8 S176
6 W5PT09 Myosin heavy chain 2 gQTVEQVtNAVGALAk T(3): 0.0; T(8): 100.0 T422
sGEGQDDAGELDFSGLLk S(1): 100.0; S(14): 0.0 S151
7 W5Q0I1 Myosin binding protein C S(3): 0.0; S(5): 0.0; S(13): 13.3;
fDScSFDLEVHESTGttPNIDIR T138,T139
T(14): 13.3; T(16): 86.7; T(17): 86.7
T(1): 0.0; S(2): 0.0; S(7): 2.2; S(8):
tSEMEASSsVR S34313
2.2; S(9): 95.6
aEFVcsISk S(6): 97.4; S(8): 2.6 S14375
8 W5Q754 Titin rsLGDIsDEELLLPIDDYLAmk S(2): 100.0; S(7): 100.0; Y(18): 0.0 S33892,S33897
vTsDNLmSR T(2): 2.3; S(3): 95.4; S(8): 2.3 S18570
T(1): 0.0; S(4): 2.1; T(5): 95.8; S(9):
tIVsTAQISETR T210
2.1; T(11): 0.0
T(2): 97.4; S(3): 2.5; T(5): 0.1; S(7):
ftSVTDsLEQVLAk T2938, S2943
9 W5PFV9 Nebulin 99.9
mVGFQsLQDDPk S(6): 100.0 S1099
S(1): 97.7; S(3): 67.3; S(4): 67.3; S230, S232, S233,
10 W5Q1Q5 Myotilin sRssSRGDmSDQDAIQEk
S(5): 67.3; S(10): 0.6 S234
aIsEELDHALNDMTsI S(3): 100.0; T(14): 50.0; S(15): 50.0 S271, T282/S283
11 B2LU28 TPM1
lEksIDDLEDELYAQk S(4): 100.0; Y(13): 0.0 S252
12 W5QFE3 Cardiac phospholamban astIEmPQQAR S(2): 100.0; T(3): 100.0 S16, T17
13 W5NU05 Myosin light chain kinase 2 iSsSGALMALGV S(2): 1.3; S(3): 97.4; S(4): 1.3 S617
rAEGANsNVFSmFEQTQIQEF S(6): 100.0; S(10): 100.0; T(15):
14 B6VCA9 Myosin light chain 2 S15,S19,T24
k 100.0
15 A9P323 Heat shock protein 90 alpha lGIHEDsQNR S(7): 100.0 S443
16 W5PDJ3 PDZ and LIM domain 3 lWsPQVTEDGk S(3): 100.0; T(7): 0.0 S89
eWYQStIPQSPsPAPDDQEEG Y(3): 0.0; S(5): 0.0; T(6): 0.0; S(10):
17 W5PBB8 Phosphodiesterase S585, S587
R 100.0; S(12): 100.0
Four and a half LIM domains 1 S(2): 1.1; S(5): 48.9; S(6): 48.9;
18 C8BKC8 hSGPsSYk S10/S11
protein Y(7): 1.1
T(3): 0.0; S(6): 0.0; S(8): 100.0;
19 W5NR35 LIM domain binding 3 dLTVDSAsPVYQAVIk S213
Y(11): 0.0
T(2): 2.7; S(3): 2.7; S(4): 47.5; S(5):
20 W5QBA6 ATRX, chromatin remodeler aTSsSNPsSPAPDWYk 47.5; S(8): 49.8; S(9): 49.8; Y(15): S1945/S1946
0.0
hAGHEDDDDGDDAVsTER S(15): 100.0; T(16): 0.0 S245
eSDsEEDEEEkEEDRssHEEAN S(2): 3.6; S(4): 96.4; S(16): 50.0;
S517, S538
EGSEEGGEGTR S(17): 50.0; S(25): 100.0; T(32): 0.0
Histidine rich calcium binding
21 W5PT08 hQGHEEEADDEDDDDIVsTE
protein S(18): 50.0; T(19): 50.0 S298, T299
HR
eEEDEDEGEENVsTEYGQQVH
S(13): 98.5; T(14): 1.5; Y(16): 0.0 S196
R
Y(1): 0.2; S(3): 99.8; S(12): 96.4;
22 W5NS29 Leiomodin 2 yEsIDEDELLAsLSAEELk S15, S24
S(14): 3.6
23 W5PIG7 Enolase 1
24 W5PQL7 Tropomyosin 2
Note: Location in peptide indicates phosphosite in peptide derived in UniProt database. F, M and S represent three groups. The colour bar represents the phosphorylation level of phosphopeptide
from Hierarchical clustering analysis. The phosphorylation levels were marked from red to green in abundance from high to low. A redder colour means a higher phosphorylation level and a
greener colour means a lower phosphorylation level of the phosphopeptides. “-” represents there is no significantly different phosphopeptide in the phosphoprotein.
Glycolysis was a dynamic physiological and developmental process. In the

glycolysis pathway, the metabolism of one molecule of glucose to two molecules
of pyruvate had a net yield of two molecules of ATP, with the concomitant
production of lactate and muscle pH decline. Protein phosphorylation was regulated
by protein kinase which transferred a phosphate group from a nucleoside triphos-
phate (usually ATP) and covalently attached it to amino acids. Phosphorylation of
pyruvate kinase could maintain high activity in low pH value. Therefore, ATP and
low pH could also affect phosphorylation.
Further research with different postmortem time will be beneficial to fully
understand the regulation of protein phosphorylation on postmortem changes and
meat quality development.
6.4 Validation of Protein Phosphorylation on Glycolysis and the Regulation. . . 109
Table 6.4 Protein identification of 11 gel bands with significant difference (Chen et al. 2019a)
Accession Sequence
No. noa Protein name Massb Scorec Matchesd coveragee
Band W5NZK9 Filamin C 278,503 5152 282(168) 59(45)
1
Band O18751 Glycogen phosphorylase 97,702 1994 188(86) 38(25)
5
Band W5QDD4 ATP-dependent 95,200 1874 123(73) 21(16)
7 6-phosphofructokinase
Band W5PJB6 Phosphoglucomutase 1 65,544 3401 188(125) 24(21)
8
Band W5QC41 Pyruvate kinase 62,180 3084 164(114) 22(19)
9
Band W5P323 Glucose-6-phosphate 63,079 3466 220(151) 16(13)
10 isomerase
Band W5PIG6 Enolase 1 49,727 1096 86(52) 15(11)
11 W5P663 Enolase 3 47,382 1006 86(44) 14(11)
Band W5P1X9 Fructose-bisphosphate 39,925 1855 122(69) 20(18)
14 aldolase
Band W5PDG3 Glyceraldehyde-3-phos- 36,241 1502 117(51) 14(9)
15 phate dehydrogenase
Band W5P5W9 Triosephosphate isomerase 23,021 2637 116(89) 10(10)
18
Band C5IJA8 Adenylate kinase isoen- 21,750 2140 142(99) 11(8)
19 zyme 1
Note: The ovine Longissimus thoracis were crushed and incubated at 4 C, and then snap frozen in
liquid nitrogen after collection at different time. The 11 bands selected were significantly different
in the phosphorylation level among the three groups
a
Accession numbers were derived from the UniProt database.
b
Theoretical molecular weight (recorded in UniProt database).
c
For the proteins identified in more than one band, the highest score was presented.
d
Number of matched peptides, total matched peptides (credible matched peptides).
e
Number of matched amino acid sequence, total matched sequence (credible matched sequence).
6.4 Validation of Protein Phosphorylation on Glycolysis

and the Regulation Mechanism of Enzyme Activity
To verify the effect of protein phosphorylation on glycolysis and elucidate the

regulatory mechanism from the perspective of enzyme activity, the ovine muscle
was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor,
and the activity of glycogen phosphorylase, pyruvate kinase, and PFK was
measured.
6.4.1 Global Phosphorylation of Sarcoplasmic Protein

with the Regulation of Kinase or Phosphatase Inhibitors
The global phosphorylation level of the sarcoplasmic protein was evaluated by

densitometry analysis of phosphoproteins and total proteins with gel stain. The
global phosphorylation level of the sarcoplasmic protein increased early PM and
then decreased (P < 0.05). These results were consistent with Huang’s data about
protein phosphorylation in muscle with intermediate pH decline rates (Huang et al.
2011). After incubation for 12 h, the phosphorylation level of the kinase inhibitor
group was significantly lower than that in the other two groups, and the phosphor-
ylation level of the phosphatase inhibitor group was significantly higher at 48 h PM
compared to the control group (P < 0.05) (Fig. 6.8).
Protein phosphorylation and dephosphorylation had diverse effects in cellular
regulation and signalling, which were regulated by the competing activities of
protein kinases and phosphatases (Manning et al. 2002). Protein kinases catalyzed
the transfer of phosphate groups from ATP to molecules, whereas phosphatases
removed phosphate groups from molecules. P0300, as a kinase inhibitor, was a
Fig. 6.8 Global phosphorylation level of sarcoplasmic protein. Different letters (x, y, z) at the same
time points represent significant difference among the three groups (P < 0.05). Different letters (A–
B) in the same group represent significant difference at different PM time (P < 0.05). (Chen et al.
2019a)
synthetic peptide used for studying the cAMP-dependent protein kinase and could
inhibit this protein kinase competitively. PhosStop Roche, used as a phosphatase
inhibitor, had a broad spectrum of action against phosphatases, inhibiting protein
phosphatases competitively. The samples were “intact” meat, which involved a
series of physic-biochemical changes. The inhibitors showed a significant difference
after 12 h of incubation, maybe because they needed time to enter the muscle cell to
function. The significant difference among the three groups revealed that the inhib-
itors were effective in modulating the level of protein phosphorylation.
6.4.2 Glycolytic Rate
The pH value and lactic acid content of the three groups were shown in Fig. 6.9.
Generally, the ovine muscle pH decreased dramatically within the first 12 h, but
remained stable afterwards. Meanwhile, the lactic acid corresponded well with the
pH value, which increased within the first 12 h but remained stable afterwards. As to
the three groups, the pH value in the phosphatase inhibitor group was significantly
lower at 2 h and 6 h than those in the control group, and the lactic acid in the
phosphatase inhibitor group was significantly higher than that in the kinase inhibitor
group during the PM time except at 6 h (p < 0.05). There was no significant
difference in pH values and lactic acid between the control and kinase inhibitor
groups throughout the whole PM period (p < 0.05).
The pH value was negatively correlated with lactate during the PM time. Lactate
was the ultimate product of anaerobic metabolism. With the formation of lactate, one
hydrogen from Nicotinamide adenine dinucleotide (NADH) and one hydrogen from
the solution were removed from the cytoplasm (Ferguson and Gerrard 2014).
Ferguson claimed there was utility in lactate formation for extending anaerobic
muscle metabolism and suggested that lactate accumulation was a good indicator
for the extent and rate of glycolysis.
Ovine muscle in the phosphatase inhibitor group with a high phosphorylation
level had higher lactate and a lower pH value compared to the control group. Li et al.
(2017) reported that the muscle of the kinase inhibitor group with a low phosphor-
ylation level had lower lactate and a higher pH value compared to the control group.
Therefore, it is logical to conclude that a high protein phosphorylation is one of the
reasons for the fast decline in the pH value.
6.4.3 Gel Band Identification
Nineteen protein bands were detected on the gels, among which 11 protein bands
that significantly differed in phosphorylation level were excised and identified
(Table 6.4). In total, 12 unique proteins were identified and most of them were
clustered into the pathways of glycogenolysis and glycolysis. They were PFK,
Fig. 6.9 pH value and lactic acid of postmortem muscle in three groups. Different letters (x, y) at
the same time points are significant difference among the three groups (p < 0.05). Different letters
(A–D) in the same group are significantly different at different PM time (p < 0.05) (Chen et al.
2019a)
Filamin C, GP, Phosphoglucomutase 1, PK, Glucose-6-phosphate isomerase,

Fructose-bisphosphate aldolase, Enolase 1, Enolase 3, Triosephosphate isomerase,
Glyceraldehyde-3-phosphate dehydrogenase, and AKI 1. Among these enzymes,
PFK and PK were rate-limiting enzymes in glycolysis, while GP catalyzed the rate-
limiting step in glycogenolysis. To explain glycolysis, the properties of glycogen
phosphorylase, PFK, and pyruvate kinase have been studied (Scheffler and Gerrard
2007). Therefore, these enzymes should be given more attention.
6.4.4 Glycolytic Enzymes Activities and Phosphorylation

Level After Regulation
6.4.4.1 Glycogen Phosphorylase
The phosphorylation level and activity of glycogen phosphorylase (GP) were shown
in Fig. 6.10. The activity of GP was basically stable during the postmortem
(PM) time. As to the three groups, the activity of GP in the phosphatase inhibitor
group was significantly higher than in the other two groups (P < 0.05). The
phosphorylation level of GP in the phosphatase inhibitor group at 2 h PM was
significantly higher than that in the control group, which was not observed at the
other PM times (P < 0.05). The correlation analysis given in Table 6.5 showed that
the activity of GP had a positive correlation with the phosphorylation level of GP
during the PM time.
GP catalyzed the breakdown of glycogen to glucose-1-phosphate for glycolysis
and was crucial in controlling glycogenolysis (Aizawa et al. 2017). GP existed in
phosphorylated GP a and dephosphorylated GP b forms. When GP b was phosphor-
ylated at serine 14, its structure changed, transforming it into the active GP a form,
which represented the first step in enzyme activation (Schwägele et al. 1996;
Johnson 1992; Sprang et al. 1988). The change in structure involved of the amino-
and carboxyl-terminal domains of GP rotating apart by 5 , which increased the
access of substrates to the catalytic site (Sprang et al. 1991). The activity of GP was
significantly higher in the phosphatase inhibitor group, theoretically, more GP a
form and a higher phosphorylation level should exist in the phosphatase inhibitor
group. Although without a statistical difference, there was a numerical difference
between the control and phosphatase inhibitor groups. The increased activity of GP
further accelerated the rate of glycogenolysis, resulting in low glycogen content and
pH value. Therefore, phosphorylation of GP was important in the regulation of
glycolysis.
6.4.4.2 Pyruvate Kinase
The phosphorylation level and the activity of pyruvate kinase (PK) was shown in
Fig. 6.11. There was no significant difference in phosphorylation level among the
40
glycogen phosphorylase activity
kinase inhibitor control phosphotase inhibitor

35
x x x
(nmol/min/mg pro)
x
30
x
y
25 y y
xy y
20 y y y y
y
15
10
5
0
0.5h 2h 6h 12h 24h 48h
Time
Fig. 6.10 Glycogen content, the phosphorylation level, and activity of glycogen phosphatase in
postmortem muscle. Different letters (x, y) at the same time points are significantly different among
three groups except at 12 h PM, where the phosphorylation level of PK in the

phosphatase inhibitor group was significantly higher than that in the control group
(P < 0.05). As for the enzymatic activity, there was a significant difference among
the three groups from 6 h onwards, with the activity in the phosphatase inhibitor
group being significantly higher than in the other two groups (P < 0.05).
PK was a critical rate-limiting enzyme that catalyzed the irreversible conversion
of phosphoenolpyruvate to pyruvate (Gupta and Bamezai 2010). It has been reported
that PK had two isoforms in muscle: isoform 2 arose from isoform 1 through
phosphorylation (Schwägele et al. 1996). Phosphorylation could cause pyruvate
kinase to retain higher activity under acidic conditions (Schwägele et al. 1996).
Heiden et al. (2010) reported that PK was found to be more active after enzyme
phosphorylation. In the phosphatase inhibitor group, the phosphorylation level and
the activity had the same trend in PM time. In terms of the three groups, the
phosphatase inhibitor group had a relatively higher phosphorylation level and
activity. Since protein phosphorylation altered the structure, activity, and stability
of the protein, it was likely that phosphorylation of PK had a positive effect on the
activity of PK.
6.4.4.3 Phosphofructokinase
The phosphorylation level and activity of PFK were shown in Fig. 6.12. There was
no significant difference in the phosphorylation level between the kinase inhibitor
and control groups PM (P < 0.05). After 24 h incubation, the phosphorylation level
of PFK in the phosphatase inhibitor group was significantly higher than that in the
control group (P < 0.05). The activity of PFK was significantly higher in the
phosphatase inhibitor group at 6 h and 12 h than in the other two groups
(P < 0.05). The activity of PFK was positively correlated with the phosphorylation
level (Table 6.5).
PFK catalyzed the conversion of fructose 6-phosphate and ATP to fructose
1,6-bisphosphate and ADP, which was the first key step in the glycolytic pathway.
Phosphorylation of PFK altered the kinetic behavior of the enzyme and regulated the
compartmentalization of the enzyme in vitro and in vivo (Luther and Lee 1986).
Some studies have demonstrated that PFK was activated after phosphorylation by
AMPK, thus promoting glycolysis (Marsin et al. 2000). The kinase inhibitor P0300
used in the present research was a specific kinase inhibitor, which inhibited the
cAMP-dependent protein kinase, specially protein kinase A, contributing no signif-
icant difference in PFK phosphorylation level between the kinase inhibitor and
control groups. AMPK could phosphorylate PFK-2 which catalyzed the formation
of fructose 2,6-bisphosphate. This product was an allosteric activator of PFK-1, a
⁄
Fig. 6.10 (continued) the three groups (P < 0.05). Different letters (A, B) in the same group are
significantly different at different PM time (P < 0.05). Adapted from (Chen et al. 2019a)
116
Table 6.5 Pearson correlation coefficients of glycolytic rate, glycolytic enzymes phosphorylation level and activities (Chen et al. 2019a)
Glycogen GP activity GP p-level PK activity PK p-level PFK activity PFK p-level Global p-level pH lactate
Glycogen 1 20.527* 0.1006 0.0577 0.4174 0.2105 0.38 20.496* 0.7199** 20.912**
0.0246 0.6912 0.82 0.0848 0.4019 0.1197 0.036 0.0008 <0.0001
GP activity 20.52704* 1 0.6326** 0.7325** 0.5842* 0.7592** 0.771** 0.588* 0.0352 0.3638
0.0246 0.0048 0.0005 0.0109 0.0003 2E-04 0.01 0.8896 0.1378
GP p-level 0.10061 0.6326** 1 0.619** 0.1543 0.5528* 0.561* 0.2009 0.5842* 0.171
0.6912 0.0048 0.0062 0.5409 0.0174 0.016 0.4241 0.0109 0.4962
PK activity 0.05773 0.7325** 0.619** 1 0.2837 0.7711** 0.594** 0.3455 0.4599 0.105
0.82 0.0005 0.0062 0.254 0.0002 0.009 0.1603 0.0548 0.6797
PK p-level 0.41739 0.5842* 0.1543 0.2837 1 0.3872 0.492* 0.492* 0.3325 0.4838
0.0848 0.0109 0.5409 0.254 0.1124 0.038 0.038 0.1776 0.0419
PFK activity 0.21046 0.7592** 0.5528* 0.7711** 0.3872 1 0.503* 0.2336 0.1623 0.0682
0.4019 0.0003 0.0174 0.0002 0.1124 0.033 0.351 0.52 0.7881
PFK p-level 0.38014 0.7709** 0.5605* 0.5938** 0.4918* 0.503* 1 0.622** 0.0353 0.3472
0.1197 0.0002 0.0155 0.0094 0.0382 0.0333 0.006 0.8895 0.158
Global p-level 20.49599* 0.5884* 0.2009 0.3455 0.4919* 0.2336 0.622** 1 0.137 0.3682
0.0363 0.0102 0.4241 0.1603 0.0381 0.351 0.006 0.5878 0.1327
pH 0.71992** 0.0352 0.5842* 0.4599 0.3325 0.1623 0.0353 0.137 1 20.848**
0.0008 0.8896 0.0109 0.0548 0.1776 0.52 0.8895 0.5878 <0.0001
Lactate 20.9123** 0.3638 0.1715 0.1046 0.4838* 0.0682 0.3472 0.3682 20.848** 1
<0.0001 0.1378 0.4962 0.6797 0.0419 0.7881 0.158 0.1327 <0.0001
Abbreviations: GP glycogen phosphorylase, PK pyruvate kinase, PFK phosphofructokinase, p-level phosphorylation level. The ovine Longissimus thoracis
were crushed and incubated at 4 C, and then snap frozen in liquid nitrogen after collection at different times. The first row of each indicator represents
correlation coefficient, and the second row represents p value. Significant levels: *0.01 <p< 0.05; **p< 0.01
6 Mechanism of the Effect of Protein Phosphorylation on Postmortem Glycolysis
50 kinase inhibitor control phosphotase inhibitor

45 x
pyruvate kinase activity (U/gprot)
x
40
x
35
x
30
y y xy
25 y y
y y y
20
15
10
5
0
0.5h 2h 6h 12h 24h 48h
Time
Fig. 6.11 The phosphorylation level and activity of pyruvate kinase in postmortem muscle.
Different letters (x, y) at the same time points are significantly different among the three groups
(P < 0.05). Different letters (A, B) in the same group are significantly different at different PM time
(P < 0.05). Adapted from (Chen et al. 2019a)
key rate-limiting enzyme of glycolysis (Scheffler and Gerrard 2007). In the present
study, the activity of PFK was positively correlated with the phosphorylation level.
Glycolysis was a sequence of enzymatic reactions that were determined by the
activity of glycolytic enzymes. GP, PK, and PFK were rate-limiting enzymes that
5.5
phosphofructokinase
4.5 kinase inhibitor control phosphotase inhibitor
phosphorylation level (P/T)
x x
3.5
xy xy
y y
2.5
1.5
0.5
-0.5 0.5h 2h 6h 12h 24h 48h

Time
22 kinase inhibitor control phosphotase inhibitor

20
x
phosphofructokinase activity
18 x
(nmol/min/mgprot)
16
14 y y
y
12
10
y
8
4
0.5h 2h 6h 12h 24h 48h
Time
Fig. 6.12 The phosphorylation level and activity of PFK in postmortem muscle. Different letters
(x, y) at the same time points are significantly different among the three groups (P < 0.05). Adapted
from (Chen et al. 2019a)
controlled the rate of PM metabolism. The activity of these enzymes was signifi-
cantly higher in the phosphatase inhibitor group with high phosphorylation level,
which revealed protein phosphorylation might play a positive role in the regulation
of glycolysis. Shen and Du (2005) reported that the glycolysis and pH decline were
References 119
indirectly affected by the phosphorylation status of AMPK in PM muscle. Therefore,

we could infer that glycolysis was indirectly affected by protein phosphorylation
through the regulation of enzyme activity.
6.5 Conclusions
The present research focused on the effects of protein phosphorylation on postmor-

tem glycolysis and glycolytic enzymes in ovine muscle. The global phosphorylation
level of sarcoplasmic proteins increased early postmortem and then decreased
afterwards in general. The results of fast, moderate, and slow glycolytic rate groups
revealed that the phosphorylation of sarcoplasmic proteins was related to muscle pH
decline at early postmortem. 24 phosphoproteins were identified to be related to the
glycolytic rate via Quantitative proteomic iTRAQ. Phosphorylation of pyruvate
kinase at Thr157, negatively correlated with the glycolytic rate, deserved more
attention in future research. To validate the effect of protein phosphorylation on
glycolysis, ovine muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a
phosphatase inhibitor, therefore resulted in different phosphorylation levels. The pH
value and lactate content revealed that a high phosphorylation level was one of the
reasons for the fast pH decline. The activity of glycogen phosphorylase, pyruvate
kinase, and PFK was positively correlated with their phosphorylation level, from
which we could infer that protein phosphorylation played a role in postmortem
glycolysis through the regulation of enzyme activity in ovine muscle. In summary,
protein phosphorylation was related to postmortem glycolysis and glycolytic
enzymes, and the phosphorylation level of glycolytic enzymes may influence its
activities, further regulating postmortem glycolysis.
Acknowledgments Parts of this chapter are reprinted from Food Chemistry, 280, Chen, L., et al.,
quantitative phosphoproteomic analysis of ovine muscle with different postmortem glycolytic rates,
203-209; Food Chemistry, 293, Chen, L., et al., Effects of protein phosphorylation on glycolysis
through the regulation of enzyme activity in ovine muscle, 537-544; International Journal of Food
Science and Technology, 53(12), Chen, L., et al., The effect of sarcoplasmic protein phosphoryla-
tion on glycolysis in postmortem ovine muscle, 2714-2722. Copyright (2020), with permission
from Elsevier.
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Chapter 7
Phosphorylation on Myofibril Protein
Degradation
Abstract Myofibrillar proteins degradation contributes to meat tenderization during

postmortem. Protein phosphorylation has been revealed to be associated with meat
tenderness in recent years. This chapter aimed to investigate the effect of myofibrillar
protein phosphorylation on the degradation susceptibility by μ-calpain. Protein
kinase A (PKA) and alkaline phosphatase (AP) were added to myofibrillar proteins,
extracted from ovine longissimus lumborum (LL) muscles, to modify the phosphor-
ylation level for 30 min at 30 C. The samples were then incubated with μ-calpain at
4 C at different Ca2+ concentrations. The results showed that more degradation
products of myosin heavy chain, actin, titin, desmin, and troponin T were detected
with the increase of Ca2+ concentration. The protein degradation was higher in AP
treated group compared to the control group. This research indicated that phosphor-
ylation prevented proteolytic susceptibility of myofibrillar proteins to degradation by
μ-calpain. Protein phosphorylation made a major contribution in meat tenderization
during postmortem.
Keywords Phosphorylation · Protein degradation · Myosin heavy chain · Actin ·

Titin · Desmin · Troponin T
7.1 Introduction
Tenderness is considered to be one of the most important quality attributes. The

degradation of myofibrillar proteins and disruption of myofibril structure by proteo-
lytic enzymes are reported to be the key mechanisms for tenderization during
postmortem (Huff Lonergan et al. 2010). The calpain system is the primary endog-
enous enzyme (Du et al. 2017). The stability and proteolysis of myofibrillar proteins
are also regulated by protein modifications. For example, oxidation and nitrosylation
affect the proteolytic activity of calpain (Li et al. 2018).
Protein phosphorylation, the most common and important post-translational
modification, has been reported to be associated with meat tenderness in recent
years (Chen et al. 2016; Gannon et al. 2008). High phosphorylation of Z-disc related
proteins has been detected in tough bovine meat by D'alessandro and Zolla (2013),

https://doi.org/10.1007/978-981-15-9441-0_7
124 7 Mechanism of the Effect of Protein Phosphorylation on Myofibril Protein. . .
who found that phosphorylation of Z-disc proteins probably increased interactions of

proteins at Z-line and reduced the accessibility of proteases, resulting in tougher
meat (D’alessandro et al. 2012a, b; D’Alessandro and Zolla 2013). In addition, Chen
et al. (2016) observed that the global phosphorylation level of tough meat was
significantly higher than tender meat at 4, 12, and 24 h postmortem. All these studies
have shown that protein phosphorylation could regulate protein stability and degra-
dation protein, which contributed to the meat tenderization. In addition, another
research founded out that phosphorylation protected proteins from calpain-mediated
proteolysis by decreasing the substrate recognition or improving the protein stability
(Zhang et al. 2012), while phosphorylation enhanced the degradation of some
protein by μ-calpain (Shen et al. 2001).
However, the mechanism by which protein phosphorylation influences meat
tenderness is still unclear, especially the impact of phosphorylation of myofibrillar
proteins on substrate recognition and degradation by μ-calpain remains unknown.
Therefore, we investigated the effect of the phosphorylation on myofibrillar protein
(myosin, action, titin, Troponin T and desmin) degradation in vitro, with the addition
of protein kinase A (PKA) and alkaline phosphatase (AP) to regulate phosphoryla-
tion level which affects degradation of these proteins. Moreover, the 3D structure of
titin fragment was simulated and molecular dynamics trajectory of the constructed
proteins was analyzed using the Discovery Studio™. The outcome of this study may
further improve our understanding of the mechanism of phosphorylation on degra-
dation of titin to regulate postmortem meat tenderization.
7.2 The Effect of Protein Phosphorylation on Myosin

and Actin Degradation
Proteolytic degradation of muscle occurs simultaneously with postmortem cell death

and meat aging, resulting in the production of protein fragments. Myosin and actin
are the first two abundant proteins in skeletal muscle and the main components of
skeletal muscle thin filaments. Protein degradation is primarily responsible for
postmortem meat tenderization, which might be affected by protein phosphorylation.
The purpose of this research was to investigate the effect of protein phosphorylation
on myosin and actin degradation during postmortem. Here, we regulated the phos-
phorylation pattern of protein by protein kinase inhibitors and phosphatase inhibi-
tors, and analyzed the effect of protein phosphorylation on myosin and actin
degradation.
7.2 The Effect of Protein Phosphorylation on Myosin and Actin Degradation 125
7.2.1 Global Phosphorylation Level of Myofibrillar Protein
The phosphorylation level of myofibrillar proteins was defined as the ratio of

phosphoproteins to total proteins. After electrophoresis, as shown in Fig. 7.1, phos-
phorylated proteins and total proteins were stained with Pro-Q Diamond and
SYPRO Ruby fluorescence staining agents, respectively. All the 21 visible myofi-
brillar protein bands were selected for total phosphorylation level quantification. The
global phosphorylation level of myofibrillar proteins significantly reduced during
storage for all the three groups (Fig. 7.2). Compared with the control group, the total
Fig. 7.1 Phosphorylation level of myofibrillar proteins (protein kinase inhibition, control, phos-
phatase inhibition, ovine longissimus). (a) Gel image of total (T) proteins stained with SYPRO®
Ruby. (b) Gel image of phosphoproteins (P) stained with Pro-Q®. Protein bands B1–B7 were
selected for LC-MS/MS identification. Protein bands 1–21 were selected for relative intensity and
phosphorylation level analysis (Li et al. 2018)
Protein kinase inhibition Control phosphatase inhibition

1.0
Phosphorylation level (P/T) a a
a X a
0.9 a b X b
X bc b
X b
0.8 b Y cd X
Y X
X de
e
c Y
0.7 X
Z cd
Y d d
0.6 Z Y
0.5
2h 1d 2d 3d 5d 7d
Storage time
Fig. 7.2 Global phosphorylation level of myofibrillar proteins. Values lacking common lowercase
letters are significantly different within the same group at different storage time points (P < 0.05).
Values lacking common capital letters are significantly different among the three groups at the same
storage time point (P < 0.05) (Li et al. 2018)
phosphorylation level of myofibrillar proteins was significantly lower in the protein

kinase inhibition group. Furthermore, the global phosphorylation level of myofibril-
lar proteins in muscle storage for 2 days and 5 days was significantly higher than that
in the control group. The results showed that the inhibitors used in the study could
effectively regulate the phosphorylation status of myofibrillar proteins in postmor-
tem muscle (Li et al. 2018).
7.2.2 Degradation of Myosin and Actin
Myosin and actin are the two most abundant proteins in the thick filament and thin
filament (Huff Lonergan et al. 2010). As shown in Fig. 7.1, several proteins
degraded during storage, and 7 protein bands were selected for LC-MS/MS identi-
fication (Table 7.1). Myosin fragment was identified in band B1, which might be
myosin heavy chain. Moreover, myosin fragments were also identified in B2 band,
which appeared during storage for 1 day in the protein kinase inhibitory group and
the control group, but not detected in the phosphatase inhibitory group. Actin was
identified in band B4, B5, B6, and B7. As shown in Fig. 7.3, myosin heavy chain and
actin were not observed with significant degradation. This was consistent with Huff
Lonergan et al. (2010), which reported no significant changes in myosin heavy chain
and actin during postmortem. However, myosin degradation fragments were iden-
tified in bands B1, B2, B3, B4 and B5, and actin degradation fragments were
identified in bands B4, B5, B6, and B7 (Table 7.1) by LC-MS/MS. This was
Table 7.1 LC-MS/MS identified proteins in 7 bands from SDS-PAGE gel (Li et al. 2018)
No. Accession Protein name Score Mass Matches Sequences Sequence coverage
B1 Q9BE40 Myosin-1 223,764 30,521 1471(942) 191(166) 67
F1MRC2 Myosin-2 224,106 27,110 1325(841) 181(155) 64
F1MJ28 Alpha-1,4 glucan phosphorylase 8215 97,674 527(317) 69(60) 70
B2 Q9BE40 Myosin-1 223,764 19,750 965(589) 169(133) 65
F1MRC2 Myosin-2 224,106 18,130 894(545) 163(123) 62
Q0IIG5 ATP-dependent 6-phosphofructokinase 86,095 7613 432(261) 39(33) 56
Q9BE39 Myosin-7 223,889 5450 347(191) 112(73) 53
B3 O62654 Desmin 8878 53,556 555(336) 61(51) 88
P19483 ATP synthase subunit alpha 2325 59,797 110(65) 30(23) 57
P48616 Vimentin 2060 53,752 162(87) 38(28) 65
Q9BE40 Myosin-1 1823 223,764 105(60) 54(35) 30
P00829 ATP synthase subunit beta 1769 56,249 108(57) 23(21) 63
F1MRC2 Myosin-2 1747 224,106 108(55) 53(33) 29
B4 P68138 Actin 42,366 9493 509(362) 33(29) 85
Q8MKI0 Troponin T fast skeletal muscle type 30,682 8488 524(328) 31(23) 64
Q8MKH9 Troponin T fast skeletal muscle type 30,684 8266 473(302) 31(22) 64
Q3ZC07 Actin 42,334 7087 446(311) 32(27) 85
Q9BE40 Myosin-1 223,764 7086 351(204) 94(57) 43
F1MRC2 Myosin-2 224,106 6917 351(200) 91(51) 41
B5 Q5KR49 Tropomyosin alpha-1 chain 32,732 11,777 726(454) 59(50) 92
7.2 The Effect of Protein Phosphorylation on Myosin and Actin Degradation
Q5KR48 Tropomyosin beta chain 32,931 9175 621(355) 58(51) 95

Q5KR48–2 Isoform 2 of Tropomyosin beta chain 33,049 8783 590(341) 53(45) 85
Q5KR47 Tropomyosin alpha-3 chain 32,856 6298 412(233) 54(41) 94
P68138 Actin 42,366 4623 266(158) 31(29) 91
(continued)
127
128
No. Accession Protein name Score Mass Matches Sequences Sequence coverage
Q9BE40 Myosin-1 223,764 3842 217(106) 74(40) 34
B6 P68138 Actin 42,366 6672 420(270) 31(28) 87
P02722 ADP/ATP translocase 1 33,174 3402 237(150) 26(24) 62
B7 A5PJM2 TNNI2 protein (fragment) 23,707 7452 669(332) 29(22) 74
P68138 Actin 42,366 1561 117(54) 25(17) 76
Q9XSC6 Creatine kinase M-type 43,190 1411 92(48) 23(12) 58
E1BEL7 Heat shock protein beta-1 22,621 1102 85(42) 15(13) 68
7 Mechanism of the Effect of Protein Phosphorylation on Myofibril Protein. . .
7.3 Phosphorylation Prevents Myosin and Actin Degradation by μ-Calpain In Vitro 129
Fig. 7.3 Immunoblot analysis of myosin heavy chain (a) and actin (c) in ovine longissimus during
storage (Li et al. 2018)
consistent with the research of Lametsch et al. (2002), which reported that myosin
and actin degradation fragments could be detected by sensitive methods (LC-MS/
MS) in postmortem muscle. In general, myosin heavy chain and actin showed
limited degradation in postmortem muscle. This study showed that dephosphoryla-
tion can partly promote the degradation of myosin heavy chain and actin, suggesting
that protein phosphorylation may play an important role in postmortem meat
tenderization.
7.3 Phosphorylation Prevents Myosin and Actin

Degradation by μ-Calpain In Vitro
Degradation of myofibrillar proteins is conducive to meat tenderization during

postmortem. Gannon et al. (2008) reported that protein phosphorylation was asso-
ciated with meat tenderness. The aim of this research was to determine the effect of
myofibrillar protein phosphorylation on the degradation sensitivity by μ-calpain.
Myofibrillar proteins were incubated with PKA or AP to increase or decrease protein
phosphorylation levels, respectively, and the effect of protein phosphorylation on
myosin and actin degradation was analyzed.
7.3.1 Phosphorylation Level of Myosin Heavy Chain

and Actin
The ratio of phosphorylated protein to total protein was used to assess protein
phosphorylation level. As shown in Fig. 7.4, the global phosphorylation level of
myofibrillar proteins in the PKA group was significantly higher than that in the
1.0
b c
0.9

0.8
0.7 a
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Control AP PKA
(C) Global phosphorylation level
(1) myosin heavy chain (2) actin

0.7 0.3
Phosphoraylation level (P/T)
Phosphoraylation level (P/T)
0.6
a a b
0.5
0.2
0.4
0.3
0.1
0.2
0.1
0.0 0.0
Control AP PKA Control AP PKA
Fig. 7.4 Assessment of the phosphorylation level of myofibril proteins (Control, AP, and PKA) by
1-DE. (a) Gel image of total (T) proteins stained with SYPRO Ruby. (b) Gel image of phospho-
proteins (P) stained with Pro-Q. (c) Global phosphorylation level (P/T). (1–2) protein phosphory-
lation level (P/T) in two individual protein bands labeled in Fig. 7.1a, (1) Band 1, mainly contains
myosin heavy chain, (2) Band 2, mainly contains actin. Values with different letters are significantly
different (P < 0.05) (Li et al. 2017)
control group. And the global phosphorylation level of myofibrillar proteins in the
AP group was significantly lower than that in the control group. These results
indicated that PKA and AP can significantly increase and decrease the overall
phosphorylation level of myofibrillar proteins in vitro, respectively. The results
also indicated that PKA and AP had an obvious regulatory effect on protein
phosphorylation in vitro.
In order to further figure out which type of protein phosphorylation was
influenced by PKA and AP, the phosphorylation level of myosin heavy chains
(MHC, band 1 in Fig. 7.4a) and actin (band 3 in Fig. 7.4a) was analyzed. As
7.3 Phosphorylation Prevents Myosin and Actin Degradation by μ-Calpain In Vitro 131
shown in Fig. 7.4, PKA and AP had no significant influence on MHC phosphory-
lation. However, PKA incubation increased the phosphorylation of actin, but AP did
not change the phosphorylation level of actin.
7.3.2 Myosin and Actin Degradation by μ-Calpain

at Different Ca2+ Concentration
In a bid to evaluate the effect of protein phosphorylation on myofibrillar protein

degradation by μ-calpain, myofibrillar proteins treated with PKA or AP were
incubated with μ-calpain at different Ca2+ concentrations.
Myosin is the most abundant protein in skeletal muscle and the main component
of muscle filaments in sarcomere. Four bands of MHC in untreated samples of
μ-calpain were detected by immunoblotting, the molecular weight of four bands
was approximately 180, 130, 110, and 70 kDa, respectively (Fig. 7.5). Approxi-
mately 95 KDa new degradation bands were detected in the control and AP group
(1 mM Ca2+) (Fig. 7.5: a3), indicating the degradation of MHC. The gray value of
band 1 in control group did not change significantly, but increased significantly in
AP group after incubation for 30 min (Fig. 7.5: a3 and b). Furthermore, the relative
gray value of band 1 in the AP group was significantly higher than that in the control
group at incubation for 5 and 30 min.
These results indicated that MHC phosphorylation by PKA prevented the hydro-
lysis of μ-calpain, but the dephosphorylation by AP promoted its degradation.
Actin is the second most abundant protein in skeletal muscle and the main
component of skeletal muscle thin filaments. Degradation of actin and myosin
disrupts the formation of actomyosin, resulting in impaired muscle structural integ-
rity (Wang et al. 2013). Degradation of actin in the PKA-treated group was also
measured. As shown in Fig. 7.6, no degradation fragments were detected in
PKA-treated group after incubation with μ-calpain at any Ca2+ concentrations used
in this study. Unlike the PKA-treated group, the control group was detected with two
actin fragments at [Ca2+] ¼ 0.1 mM, while three degradation fragments were
detected at [Ca2+] ¼ 1.0 mM. Three actin fragments were detected in the AP
treatment group at 0.1 mM and 1.0 mM Ca2+. At [Ca2+] ¼ 1.0 mM. Quantitative
analysis of actin fragments showed that the density of these three bands increased
with incubation time. The fragments of the three bands in the AP treatment group
were higher than that in the control group, indicating that the degradation of actin
increased after incubation with AP. These results showed that PKA and AP
pretreatment reduced and increased actin degradation by μ-calpain, respectively.
SDS-PAGE analysis showed that MHC and actin did not significantly degrade after
incubation with μ-calpain, but immunoblotting identified degradation products with
lower molecular weight in new bands, demonstrating that they can be hydrolyzed by
（b）band 3 （1 mM)
2
* **
volume (104)
1
Control
AP
0
5 10 30
Incubation time (min)
Fig. 7.5 Analysis of the degradation of myosin heavy chain after incubation with μ-calpain for
30 min. (a1–3) Western blot analysis of myosin heavy chain after myofibrillar proteins were
incubated with μ-calpain for 30 min at 0.01, 0.1, and 1 mM Ca2+. (b) Densitometric analysis of
band 1 in (a3). Values with/show significant difference between Control and AP group in the same
incubation time (P < 0.05); values with different letters are significantly different within the AP
group at different incubation time (P < 0.05) (Li et al. 2017)
μ-calpain. MHC and actin did not change significantly during postmortem.
However, scholars have reported that they were all substrates of μ-calpain, and
two protein degradation products were detected in postmortem muscle (Lametsch
et al. 2002; Lametsch et al. 2004).
7.4 Influence Mechanism of Protein Phosphorylation on Titin Degradation 133
(b1) bind 1 (1 mM) (b2) bind 2 (1 mM) (b3) bind 3 (1 mM)

Control AP Control AP Control AP
*ab *c *b
3
6
*a B
b
6
* *b
*b
volume (104)
5
*
volume (104)
5
volume (104)
A 2 a
4 A 4 B
a
3 3 B A
A
2 2 1
A A
1 1
0 0 0
5 10 30 5 10 30 5 10 30
Incubation time (min) Incubation time (min) Incubation time (min)
Fig. 7.6 Analysis of the degradation of actin after incubation with μ-calpain for 30 min. (a1–3)
Western blot analysis of actin after myofibrillar proteins were incubated with μ-calpain for 30 min at
0.01, 0.1, and 1 mM Ca2+. (b1–3) Densitometric analysis of band 1–3 in (a3). Values with/show
significant difference between Control and AP group at the same incubation time (P < 0.05); values
with different letters are significantly different within the same group at different incubation time
(P < 0.05) (Li et al. 2017)
7.4 Influence Mechanism of Protein Phosphorylation

on Titin Degradation
Titin is the third most abundant protein in muscle that spans half of a sarcomere with
its C-terminal end localizing in the M-line and the N-terminal forming an integral
part of Z-line in striated muscles. Its degradation weakens the longitudinal structure
of myofibrillar sarcomere and the integrity of muscle. The objective of this part was
to investigate the degradation of titin and its phosphorylation level in three positions
of sheep muscles. The pH of longissimus lumborum (LL), semimembranosus (SM),
and psoas major (PM) muscles were measured at 30 min, 1, 2, 7, 14, 21, and 28days
postmortem. Myofibrillar proteins were extracted, separated by SDS-PAGE, and
quantified by phosphor-specific staining. Phosphorylation of titin was evaluated by
Pro-Q Diamond-SYPRO Ruby staining.
7.4.1 Changes in pH Value of the Three Ovine Muscles
Muscle pH values were shown in Fig. 7.7. The pH of all the three muscles decreased
rapidly within 1 day postmortem (P < 0.05) and then did not change afterwards. The
pH of the PM muscle was significantly lower than that of the other two muscles at
0.5 h postmortem, but higher for the rest time (P < 0.05).
The pH decline was majorly due to the accumulation of lactate produced through
glycolysis during postmortem. Bouton et al. (1971) reported that pHu (ultimate pH)
in ovine muscles was achieved on the third day postmortem at 1–2 C. In the present
research, all the three muscles reached pHu after 1 day postmortem. It might because
of vacuum packing, which resulted in the quick accumulation of lactate
(Napravnikova et al. 2002). In addition, pHu of the PM muscle was significantly
higher than that of the LL and SM muscles (P < 0.05). This might be caused by the
various amount of glycogen, which determined pHu (Ferguson et al. 2008; Honikel
2014a, b).
7.0 ax
LL SM PM
6.8
6.6 ay
6.4
6.2
pH
az
6.0
bx bcx
5.8 by by bcycdx cdx bcd dex ex
bcy bcy by cdy dy dy
5.6 cdy y dy
5.4
5.2
5.0
0.5h 1d 2d 7d 14d 21d 28d
Post-mortem time
Fig. 7.7 pH values of LL, SM, and PM muscles stored at 4 C for 28 days. Values with x-y-z differ
among the three muscle types (P < 0.05). Values with a-b-c are significantly different among aging
time points within the same group (P < 0.05) (Wang et al. 2018)
7.4 Influence Mechanism of Protein Phosphorylation on Titin Degradation 135
7.4.2 Changes in Phosphorylation Level of Titin
Phosphorylation of titin was evaluated by SDS-PAGE and fluorescence staining. As

shown in Fig. 7.8, the phosphorylation level of titin from PM muscle was signifi-
cantly higher at 0.5 h, 1 and 2 days postmortem than that from the same muscle in
other time points (P < 0.05). The phosphorylation of titin from LD and SM muscles
was higher at 1 day postmortem than that of LD and SM at 7, 14, 21, and 28 days
postmortem (P < 0.05). After 2 days postmortem, the phosphorylation level of titin
from all muscles decreased. The results may be caused by the content of ATP in cold
(2–4 C) stored muscles. Protein phosphorylation was a common post-translational
modification.
Kinases catalyze phosphorylation of proteins, and this reaction occurs almost
exclusively with the existence of ATP (Macek et al. 2009). Mattio et al. (1992) found
out that a maximum content of ATP occurred 6 h after death, and then, there were no
significant difference in the slopes of ATP decrease during 1–2 days postmortem.
Finally, ATP showed a decreasing tendency after 3 days postmortem. With the
exhaustion of ATP, accumulation of different metabolic by-products and the devel-
opment of rigor mortis, the phosphorylation status of myofibrillar proteins from three
muscles (LL, SM, and PM) would be changed with postmortem time (Huang et al.
2012). In addition, the phosphorylation of titin from PM muscle was significantly
higher than that of the SM and LL muscles within 14 days postmortem (P < 0.05),
with LL muscle being lowest in titin phosphorylation. The reason might be that there
was a higher content of ATP in the PM muscles. Linear fitting analysis has been
conducted for comparison of the rate of phosphorylation level of titin decrease.
Fitting functions were shown in Fig. 7.8f. The absolute value of the slope of the
equation from PM (jKPMj ¼ 0.2476) was higher than the other two absolute values
of the slopes. The result indicated titin from PM muscles degraded faster.
7.4.3 Degradation of Titin
Titin is the largest protein in nature with a molecular weight approximately of

3000 kDa (Labeit et al. 1990). When titin is degraded, a major degradation product
is observed that migrates only slightly faster under SDS-PAGE conditions than
intact titin. This product migrates at approximately 2400 kDa (T1) (Kurzban and
Wang 1988; Huff-Lonergan et al. 2010). Another titin degradation product that has
been observed migrates at approximately 1200 kDa (T2) by SDS-PAGE analysis
(Huff-Lonergan et al. 2010). Titin from PM muscle migrated as three closely spaced
bands (T1, T1–2, T2, in the increasing order of migration) (Fig. 7.8b), which
matched with the results of Huff-Lonergan et al. (1996). Intact titin (T1) decreased
and degraded titin (T2) increased in three samples with postmortem time. However,
there were two bands (T1, T1–2) from PM muscles at 0.5 h postmortem, while there
(E)
LL SM PM
2.0
Phosphorylation leve of titin
1.8
1.6 ax
ax
1.4
ax
1.2
1.0 bx
0.8
0.6 ay bcx
aby
0.4 abcy bcy dx
cdy
ay cdx dx dx
0.2 aby aby by bz by bx
0.0
0.5h 1d 2d 7d 14d 21d 28d
Post-mortem time
LL SM PM
(F)
1.8 LL SM PM
Phosphorylation level of titin
1.6 yLL = -0.0293x + 0.2066

1.4 ax R² = 0.7014
ax
ax ySM = -0.0506x + 0.4602
1.2
R² = 0.9265
1.0
bx yPM= -0.2476x + 1.8257
0.8 R² = 0.847
0.6 ay bcx
aby
0.4 abcy bcy dx
ay cdy
0.2 aby cdx dx dx
aby by bz by bx
0.0
0.5h 1d 2d 7d 14d 21d 28d
Post-mortem time
Fig. 7.8 (a) Gel images of phosphoproteins and total proteins and quantification of protein
phosphorylation level of titin from three ovine muscles stored in refrigerator for 28 days. (a) and
(b) Gel images of phosphoproteins and total proteins from PM muscle. (a) and (c) Gels stained with
7.5 Effects of Phosphorylation on Titin Degradation at Different Ca2+. . . 137
was only one band (T1) from LL and SM muscles at the same time (Fig. 7.8d). The
rate of conversion of T1 to T2 was faster in PM muscle than in LL and SM muscles.
The slope of fitting functions (Fig. 7.8f) also indicated the rate of titin from PM
degraded faster. This was consistent with literature in which Melody et al. (2004)
reported that the degradation of titin occurred earlier in the PM than in the other two
muscles. The degradation of beef large structural proteins, including titin, was
associated with inherent muscles pHu (Wu et al. 2014a, b). High-pHu (6.2) muscles
had faster rate of titin degradation than muscles of lower pHu (5.79). In this study,
the fastest degradation of titin was detected in PM muscle of higher pH compared to
that of LL and SM muscles. The reason might be that μ-calpain was most active near
neutral pH (Koohmaraie and Geesink 2006). Higher pHu could lead to faster
autolysis of the enzyme to the 76 kDa isoform and thus high l-calpain activity in
muscle. In addition, Fig. 7.8e showed that the phosphorylation level of titin in PM,
which had the faster degradation of titin, was determined to be higher than in the
other two muscles. The reason might be that the phosphorylation of titin had a
positive effect on its degradation. Titin has been known to be phosphorylatable and
contained phosphorylation sites (Gautel et al. 2001). Protein phosphorylation was a
common post-translational modification in regulating the structure and function of
protein. The described series of steps were progressive phosphorylation, the struc-
tural change and then degradation (Chiu et al. 2008). The PEST sequence was rich in
proline (P), glutamic acid (E), serine (S), and threonine (T). The amino acid
sequences appeared to be signals for degradation. After being phosphorylated, the
structure of the protein may change. The hidden PEST may be unfolded. The
inferred conformational change allowed the casein kinases to access a previous
titin easily and led to the degradation of protein.
7.5 Effects of Phosphorylation on Titin Degradation

at Different Ca2+ Concentrations Incubation In Vitro
Titin was extracted from ovine longissimus lumborum muscles. PKA and AP were
individually added to titin in vitro to regulate its phosphorylation level. The samples
were then incubated with μ-calpain at 4 C for 2 days at Ca2+ concentrations of either
0.05 or 2 mmol/L. The pH of the systems was measured and the level of phosphor-
ylation and degradation of titin were determined.
⁄
Fig. 7.8 (continued) Pro-Q Diamond. (b) and (d) Gels stained with SYPRO Ruby. (f) Linear fitting
analysis for protein phosphorylation level of titin. Values with x-y-z differ among the three muscle
types (P < 0.05). Values with a-b-c are significantly different among aging time points within the
same group (P < 0.05) (Wang et al. 2018)
7.5.1 Phosphorylation and Dephosphorylation of Titin
The effects of PKA and AP on the phosphorylation of titin were determined

(Fig. 7.9). The bands of titin were shown on Pro-Q Diamond (Figure 7.9a) and
SYPRO Ruby stained gels (Figure 7.9b). The phosphorylation level of titin from the
PKA group, control group, and AP group was shown in Figure 7.9c. The result
showed that the phosphorylation level of titin from the PKA group was significantly
higher than that in the control group, which had a higher phosphorylation level than
the AP group (P < 0.05). It indicated that the phosphorylation of titin in vitro was
effectively altered by the treating of PKA and AP. Skeletal titin contained the N2
region (Tskhovrebova and Trinick 2003). The kinase domain in the sequence of titin
and potential phosphorylation site of titin could be phosphorylated by PKA (Gautel
et al. 1993; Sebestyen et al. 1995). In addition, the phosphorylation level of titin from
the AP group was the lowest among the three groups (Figure 7.9c, P < 0.05). This
may due to the significant effect of AP on titin. AP was a phosphohydrolase that
could catalyze the hydrolysis of monophosphate to release free inorganic phosphate
(C)
3.50
Relative phosphorylation level of titin
x
3.00
y
2.50
z
2.00
1.50
1.00
0.50
0.00
PKA Group Control Group AP Group
Fig. 7.9 Phosphorylation levels of titin from PKA group, Control group, and AP group. (a) Image
of gel stained with Pro-Q Diamond. (b) Image of gel stained with SYPRO Ruby. (c) Quantification
of phosphorylation level of titin. Values with different letters differ among three groups (P < 0.05)
(Wang et al. 2019)
7.5 Effects of Phosphorylation on Titin Degradation at Different Ca2+. . . 139
or to transfer the phosphoryl group to other alcohols (Deng et al. 2015; Nguyen et al.
2017). AP not only inhibited the phosphorylation of the kinase domain of titin, but
also promoted the dephosphorylated of other domains in the present research.
7.5.2 pH Values of Incubation Systems
The pH values of reaction systems before and after incubation were shown in
Table 7.2. The results showed that the pH value was lower after incubation than
before in each group (P < 0.05). In addition, there was no significant difference of
the pH among three treatments in this study (Table 7.2, P > 0.05). Many previous
studies have proved that pH played the vital role in the activity of μ-calpain (Du et al.
2017; Lomiwes et al. 2014; Pulford et al. 2009). The result of this research excluded
the possible different activity of μ-calpain caused by pH value.
7.5.3 Degradation of Titin
When intact titin (the T1 band) was degraded, a major degradation product T2
(approximately 2400 kDa) and another degradation product (about 1200 kDa)
were observed by using SDS-PAGE analysis Huff-Lonergan et al. 1995; Huff
Lonergan et al. 2010). Degradation of titin from the PKA group, control group,
and AP group at different concentrations of Ca2+ were present in Fig. 7.10. At the
Ca2+ concentrations of 0.05 mM, T1 and T2 bands were detected in the PKA and
control groups (Fig. 7.10a), while besides T1 and T2 bands, there was another band
weighted 1200 kDa at the Ca2+ concentration of 2 mM (Fig. 7.10b). The band
weighted 1200 kDa after 12 h incubation occurred at the Ca2+ concentration of
0.05 mM (Fig. 7.10b), while occurred at 0.5 h at the Ca2+ concentration of 2 mM in
AP group (Fig. 7.10). The degradation rate of titin became faster as the Ca2+
concentration increased (Fig. 7.10). Titin was the substrate of μ-calpain (Wu et al.
2014a, b), which was a calcium dependent protease and required micro-molar of
calcium for its activity (Biswas et al. 2016). The increased concentration of Ca2+
promoted the degradation of μ-calpain and activated μ-calpain at the same time
(Du et al. 2017).
Table 7.2 Changes in pH of Treatment pH(before) pH(after)

incubated system before and
PKA group 6.85 0.02ax 6.72 0.01bx
after incubation. (Wang et al.
2019) Control group 6.82 0.02ax 6.73 0.03bx
AP group 6.81 0.02ax 6.72 0.02bx
Notes: Values with x-y-z differ among three groups at the same
aging time (P < 0.05). Values with a-b are significantly different
among aging time points within group (P < 0.05)
Fig. 7.10 Degradation of titin from PKA group, Control group, and AP group at different Ca2+
concentrations (Wang et al. 2019)
This present study also found out that the band with a molecular weight of
1200 kDa was obtained at the Ca2+ concentrations of 0.05 in the AP group, while
it was not detected in the PKA and control groups (Fig. 7.10a, b). At the Ca2+
concentration of 2 mM, the band weighted 1200 kDa showed up in all three groups.
However, there was the band weighted 1200 kDa in the AP group at 0.5 h incuba-
tion, at 12 h incubation in the PKA group and control group (Fig. 7.10b). The results
indicated that titin in the AP group degraded faster than that in the PKA and control
groups at the same Ca2+ concentration. Previous studies reported that pH of muscle
affected the degradation of titin. In their research, they discovered that titin degraded
faster in the meat muscles with the high ultimate pH, which had a higher activity of
μ-calpain (Lomiwes et al. 2014; Wu et al. 2014a, b). However, there was no
significant difference in pH among three groups in this study (Table 7.2,
P < 0.05). It indicated that μ-calpain activity was not differently affected by pH in
the three treatments (Du et al. 2017). In the present work, titin degraded faster in the
AP group which had a lowest relative phosphorylation level (Figs. 7.9 and 7.10,
P < 0.05). The result demonstrated the dephosphorylation of titin caused by AP
accelerated the degradation of titin. The effect of AP may release free inorganic
phosphate or transfer the phosphoryl group to cause the change of structure of titin. It
was likely that dephosphorylation of titin influenced the stability of the protein.
However, the structure of the dephosphorylated titin was not measured in the present
study. Therefore, how the stability dephosphorylation of titin in vitro contributes to
the degradation of titin needs further research.
7.6 Influence of Protein Phosphorylation on Other Myofibril Proteins Degradation 141
7.6 Influence of Protein Phosphorylation on Other

Myofibril Proteins Degradation
Previous researches have reported that the degradation of Troponin T (TnT) and
desmin contributed to the meat tenderization. Degradation of TnT has been proved
be highly related to the postmortem tenderization of meat (Anderson et al. 2012;
Cruzen et al. 2014). Desmin is associated with Z-disc to connect Z-disc with other
cytoskeletal elements. Desmin degrades rapidly during postmortem meat maturation
and serves as an indicator of overall postmortem proteolysis (Huff-Lonergan et al.
2010). Further, we found out Tnt and desmin could also be phosphorylated (Li et al.
2017).
7.6.1 Troponin T (TnT)
A main TnT degradation product (30 kDa, band 2) was detected for control and AP
samples at both [Ca2+] ¼ 0.1 and 1.0 mM, which was only detected in PKA samples
at the highest Ca2+ concentration used in this study (Fig. 7.11: a2 and a3). In
addition, two other products less in abundance were also detected in this research.
Quantification of intact TnT showed that PKA-treated samples contained the most
unchanged TnT among the three treatments at any incubation time (Fig. 7.11: b1 and
b2). When comparing the control and AP treatments, less or the same amount of
intact TnT was measured in AP samples. All these data showed that increased
phosphorylation of TnT by PKA prevented its degradation, while dephosphorylation
by AP increased its susceptibility to μ-calpain.
After incubation with μ-calpain, all desmin and TnT were degraded in AP and
control samples at [Ca2+] 0.1 mM. Pretreatment of proteins with PKA reduced, while
AP increased the degradation of all the four proteins by μ-calpain. In particular, the
phosphorylation level of TnT showed a good inverse relationship with the degrada-
tion of the proteins, suggesting that phosphorylation may play an important role in
protein degradation. High phosphorylation of Z-disc related proteins, including titin
and TnT, were detected in tough bovine meat. Phosphorylation of Z-disc protein
probably led to increased interactions of myotilin, myozenin 1, and troponin at
Z-discs and reduced accessibility by proteases, resulting in tougher meat
(D’Alessandro and Zolla 2013; D’Alessandro et al. 2012a, b). In addition, higher
level of phosphorylation of structural proteins was related to lower myofibrillar
degradation index in the Longissimus dorsi of Chianina and Maremmana cattle
(D’Alessandro et al. 2012a, b). Troponin I with lower phosphorylation was more
susceptible to degradation by calpain compared with Troponin I with higher phos-
phorylation (Gomes et al. 2005), which was consistent with our study. Another
possibility was that phosphorylation may change the conformation of the protein,
leading to changed stability (Tornavaca et al. 2011) (Fig. 7.11).
Fig. 7.11 Analysis of the degradation of troponin T after incubation with μ-calpain for 30 min.
(a1–4) Western blot analysis of troponin T after myofibrillar proteins were incubated with μ-calpain
for 30 min at 0.01, 0.1 and 1 mM Ca2+. (b1) Densitometric analysis of troponin T in (a2). (b2)
7.6 Influence of Protein Phosphorylation on Other Myofibril Proteins Degradation 143
7.6.2 Desmin
Degradation of desmin by μ-calpain was shown in Fig. 7.12. Before incubation with
μ-calpain, two bands of intact desmin were detected in isolated myofibrillar proteins.
Almost all desmin was degraded and no intact desmin was detectable in control and
AP samples after incubation with μ-calpain when [Ca2+] increased to 0.1 mM. Three
bands were detected for desmin hydrolysis. Different with the control and AP
treatments, desmin in the PKA-treated samples was unchanged and no fragments
were detected (Fig. 7.12: a2), indicating the increased stability of PKA phosphory-
lated desmin. When [Ca2+] was further increased to 1.0 mM, only one band of
desmin fragment (band 3) was visible in control and AP samples. The other
two bands (band 1 and 2) were probably further digested to smaller fragments
that could not be detected by the anti-desmin antibody used in this study
(Fig. 7.12: a3). The increased proteolysis of desmin was also measured in the
PKA sample at [Ca2+] ¼ 1.0 mM, for which three products were determined. To
compare the difference between control and AP samples, band 3 was quantified by
densitometric analysis. As shown in Fig. 7.12, the relative density of band 3 was
lower for AP samples than for control, showing relatively higher level of protein
degradation in AP samples. All these data showed that dephosphorylation of desmin
by AP enhanced its degradation, while phosphorylation by PKA reduced its degra-
dation by μ-calpain.
Two isoforms of desmin (54 and 52 kDa) and three degraded fragments (50, 47
and 39 kDa) were reported in a previous study (Muroya et al. 2010). Desmin was
susceptible to proteolysis and purified desmin was thoroughly degraded after incu-
bation with μ-calpain for 1 h at 25 C (Baron et al. 2004). Consistent with the
literature, two bands of desmin and three degradation products were determined in
this study. Also, the intact desmin was thoroughly degraded in control and AP
samples at 0.1 and 1 mM Ca2+.
Structural studies have confirmed that phosphorylation induced conformational
changes in peptides corresponding to the hinge region (Penrose et al. 2004). Kim
reported that the conformation of C/EBPb changed after dual phosphorylation,
which facilitated dimerisation through its leucine zipper domains, leading to reduced
degradation (Kim et al. 2007).
⁄
Fig. 7.11 (continued) Densitometric analysis of troponin T in (a3). /means significant difference
between treatments at the same incubation time (P < 0.05); values with different letters are
significantly different within the same group at different incubation time (p < 0.05) (Li et al. 2017)
Fig. 7.12 Analysis of the degradation of desmin after incubation with μ-calpain for 30 min. (a1–3)
Western blot analysis of desmin after myofibrillar proteins were incubated with μ-calpain for 30 min
at 0.01, 0.1 and 1 mM Ca2+. (b1) Densitometric analysis of band 3 in (a2). (b2) Densitometric
analysis of band 3 in (a3). Values with/show significant difference between Control and AP group
at the same incubation time (P < 0.05); values with different letters are significantly different within
the Control group at different incubation time (P < 0.05) (Li et al. 2017)
7.7 Conclusions
PKA and AP can effectively alter the phosphorylation of isolated myofibrillar pro-
teins and individual proteins, such as titin, desmin, and TnT. The phosphorylation of
isolated proteins was significantly decreased by AP and increased proteolysis of the
three proteins by μ-calpain. In this present study, we used DS software to carry out
the simulation and make the molecular dynamics trajectory analysis in order to give
References 145
the hypothesis. The hypothesis was that AP changed the dihedral angle of serine
small and made the structure of titin unstable to promote its degradation. Additional
research is needed to determine the structural changes of the intact titin after
dephosphorylation to verify the hypothesis. The outcome of this study may improve
our understanding of the mechanism of phosphorylation on degradation of myofi-
brillar proteins to regulate postmortem meat tenderization.
Acknowledgments Parts of this chapter are reprinted from Food Chemistry, 245, Li, Z., et al.,
Dephosphorylation enhances postmortem degradation of myofibrillar proteins. Food Chemistry,
233–239; Food Chemistry, 218, Li, Z., et al., Phosphorylation prevents in vitro myofibrillar proteins
degradation by μ-calpain. Food Chemistry, 455–462; International Journal of Food Science and
Technology, 53, Wang, Y., et al., Changes in phosphorylation levels and degradation of titin in
three ovine muscles during postmortem aging. 913–920, Copyright (2020), with permission from
Elsevier. Parts of this chapter are translated from Food Science, 40, Wang, Y., et al., Effects of
Phosphorylation on titin Degradation at different Ca2+ concentrations in Vitro (Chinese). 52–57.
Copyright (2020), with permission from Journal of Food Science.
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Chapter 8
Phosphorylation on Calpain Activity
Abstract μ-Calpain is the key enzyme in postmortem meat tenderization. Studies

have shown that μ-calpain can be phosphorylated and phosphorylation influences the
activity of μ-calpain. However, whether μ-calpain can be phosphorylated in post-
mortem muscles and the possible relative influencing mechanism remains unknown.
The longissimus lumborum (LL) muscles were used in this research. The relation-
ship between phosphorylation level of sarcoplasmic proteins, μ-calpain activity, and
calpastatin degradation in mutton with different tenderness was analyzed. Alkaline
phosphatase and phosphatase inhibitor were used to modulate the phosphorylation
level of sarcoplasmic proteins, and the effect of phosphorylation of sarcoplasmic
proteins on μ-calpain activity was studied. The phosphorylation level of μ-calpain
was regulated in vitro, and the influence of dephosphorylation and phosphorylation
of μ-calpain on μ-calpain activity was investigated. The changes in the secondary
structure of μ-calpain were studied and the phosphorylation sites of μ-calpain were
identified to illustrate the directly regulatory mechanism of μ-calpain activity by
phosphorylation. The effects of calpastatin on phosphorylated μ-calpain and the
effects of phosphorylated calpastatin on μ-calpain were also analyzed. The study of
the effects of phosphorylation on the interaction between calpastatin and μ-calpain
clarified the indirectly regulatory mechanism of phosphorylation on μ-calpain
activity.
Keywords Tenderness · μ-calpain · Calpastatin · Phosphorylation ·

Dephosphorylation
8.1 Introduction
Tenderness is an important sensory quality attribute of meat, especially for beef and
lamb. The calpain system, which includes μ-calpain, m-calpain, and calpastatin, is
considered to be primarily responsible for the postmortem proteolytic tenderization
of meat (Pomponio and Ertbjerg 2012; Kemp et al. 2010). The μ-calpain is a
heterodimer composed of two subunits of 80 kDa and 28 kDa. The 80 kDa subunit
degrades to a 76 kDa peptide through a 78 kDa intermediate by autolysis for its

https://doi.org/10.1007/978-981-15-9441-0_8
150 8 Mechanism of the Effect of Protein Phosphorylation on Calpain Activity
activation (Goll et al. 2003). In addition, the activity of μ-calpain is regulated by

many factors, such as Ca2+, temperature, calpastatin, etc. (Koohmaraie 1992;
Geesink et al. 2006).
The calpains are calcium activated cysteine proteases. The 80 kDa subunit has EF
hand domains which bind Ca2+ to activate calpain. Therefore, the concentration of
Ca2+ in sarcoplasm directly influences μ-calpain activation (Goll et al. 2003; Ohno
et al. 1986). The μ-calpain is very sensitive to temperature. In postmortem bovine
muscle, μ-calpain activity is primarily regulated by muscle temperature (Mohrhauser
et al. 2014). In addition, accelerated μ-calpain activation is detected in both lamb and
beef when muscles are incubated at elevated temperatures (Geesink et al. 2000;
Hwang et al. 2004; Pomponio and Ertbjerg 2012). As one of the well-characterized
parts in calpain system, the endogenous calpastatin is the only known protein
inhibitor specific for μ-calpain and m-calpain (Crawford 1990). Each calpastatin
can inhibit four calpain molecules to limit postmortem muscle proteolysis and meat
tenderization (Goll et al. 2003; Kent et al. 2004).
Protein phosphorylation, one of the most common post-translational modifica-
tions, has intrigued scientists’ great interest in recent years for its regulation of
postmortem energy metabolism and probably meat quality (Huang et al. 2012). It
is also reported that phosphorylation of both sarcoplasmic and myofibrillar proteins
in beef carcass is altered by electrical stimulation (Li et al. 2015), indicating protein
phosphorylation may be related to meat tenderness. However, the exact biochemical
mechanism is not well understood. Biomedical studies reveal that μ-calpain is
phosphorylated by protein kinase A (PKA) and protein kinase C (PKC) (Storr
et al. 2011). There are nine phosphorylation sites in the four domains of the
80 kDa subunit of μ-calpain, including four Ser residues, three Thr residues, and
two Tyr residues (Vazquez et al. 2008). Phosphorylation of μ-calpain alters its
proteolysis ability and the location within cells (Vazquez et al. 2008; Xu and Deng
2006a, b). However, whether the μ-calpain function affected by phosphorylation in
postmortem muscle remains unknown.
8.2 Relationship Between Protein Phosphorylation

and Calpain Activity
Muscle samples were grouped into three according to myofibril fragmentation index
(MFI) at 24 h and 48 h postmortem. The relationship between phosphorylation level
of sarcoplasmic proteins, μ-calpain activity, and calpastatin degradation in postmor-
tem mutton with different tenderness was investigated. In the present study, the MFI
of 50 sheep at 24 h and 48 h postmortem was detected and the results were ranked.
Sheep with highest, middle, and lowest (5 of each) MFI value were chosen as tender,
middle, and tough groups. The results showed that there was a significant difference
between the three groups, which could be used for further analysis.
8.2 Relationship Between Protein Phosphorylation and Calpain Activity 151
8.2.1 Changes in pH in Mutton with Different Tenderness

at Postmortem
The changes in pH values in the three groups were consistent during postmor-
tem (Fig. 8.1). There was no significant difference in pH values among the three
groups at each time point (P > 0.05).
The decline rate of pH during postmortem affected meat tenderness (Marsh et al.
1987). pH was also an important factor that influences the activity and autolysis
degradation of μ-calpain (Maddock et al. 2005). Many studies have shown that the
inactivation rate of μ-calpain was faster in muscle with faster pH decline rate at
postmortem (Hwang and Thompson 2001; Rhee et al. 2006; Whipple et al. 1990).
Muscle with low pH presented low μ-calpain activity (Claeys et al. 2001). In this
study, there was no significant difference in pH value in three groups at each time
point, indicating that the activity of μ-calpain and meat tenderness was not affected
by pH value.
8.2.2 Phosphorylation Level of Sarcoplasmic Proteins During

Postmortem
The relative phosphorylation level of sarcoplasmic proteins in tender, middle, and

tough groups during postmortem is shown in Fig. 8.2. The changes in phosphory-
lation level of sarcoplasmic proteins in three groups were consistent, but tender
7.5
Tender group
Middle group
7.0
Tough group
6.5
pH
6.0
5.5
5.0
30min 1h 2h 6h 12h 24h 48h 72h 5d 7d
Postmortem time
Fig. 8.1 Changes in pH in mutton with different tenderness at postmortem (Du 2018)
1.4 a a a
a a
a
Relative phosphorylation level
1.2 a a b a
a b b b b
b c c bb b
a c b
1.0 c b b
c c
b Tender group
0.8
Middle group
0.6
Tough group
0.4
0.2
0.0
30min 1h 2h 6h 12h 24h 48h 72h 5d 7d
postmortem time
Fig. 8.2 Phosphorylation level of sarcoplasmic proteins during postmortem (Du 2018)
group had a significantly lower phosphorylation level than middle group, than that of
the tough group (P < 0.05).
Protein phosphorylation, one of the most common post-translational modifica-
tions, has intrigued scientists’ great interest in recent years for its regulation of
postmortem energy metabolism and probably meat quality (Huang et al. 2012). It
is also reported that phosphorylation of both sarcoplasmic and myofibrillar proteins
in beef carcass is altered by electrical stimulation (Li et al. 2015), indicating protein
phosphorylation may be related to meat tenderness. Our team has studied the
phosphorylation level of proteins in mutton with different tenderness and found
out that tender meat showed lower phosphorylation level of both sarcoplasmic and
myofibrillar protein, which was the same as the present study. Phosphorylation may
affect meat tenderness by influencing rigor mortis and glycolysis (Chen et al. 2016).
8.2.3 Casein Zymography Analysis of μ-/m-Calpain
In the detection of μ-calpain and m-calpain by casein zymography in the present

study, casein gels were incubated in the presence of 4 mM Ca2+. This concentration
of Ca2+ can activate both μ-calpain and m-calpain to degrade casein at their locations
in the gel. Thus, the content of detected μ-calpain and m-calpain remained proteol-
ysis ability. The results showed that the content of unautolyzed μ-calpain in all three
groups decreased during postmortem (Fig. 8.3). The proteolysis activity of
unautolyzed μ-calpain in tender group decreased faster at 6 h postmortem. At 24 h
postmortem, μ-calpain in tender group autolyzed totally and deactivated at 72 h
postmortem. In the middle group, μ-calpain deactivated at 5 d postmortem, but still
remained activity at 7 d in the tough group. During postmortem, the proteolysis
8.2 Relationship Between Protein Phosphorylation and Calpain Activity 153
Tender group Middle group Tough group

St 1 2 3 4 5 6 7 8 9 10 St 1 2 3 4 5 6 7 8 9 10 St 1 2 3 4 5 6 7 8 9 10
μ-calpain
m-calpain
Fig. 8.3 Casein zymography of μ-/m-calpain. Sarcoplasmic protein of middle group at 30 min
postmortem was regard as St. (Du 2018)
activity of m-calpain did not change in three groups. M-Calpain did not autolyze
during 7 d aging.
The content of μ-calpain in tender, middle, and tough groups decreased during
postmortem aging, indicating that μ-calpain was responsible for meat tenderization
by autolysis and degradation. Studies have shown that the proteolysis ability of
μ-calpain mainly concentrated on the first 3 days after slaughter. μ-calpain activated
because of its self-autolysis and deactivated after degrading substrate proteins. In
most cases, the concentration of Ca2+ in postmortem muscle was too low to activate
m-calpain, thus, m-calpain might not have an effect on meat tenderization postmor-
tem (Koohmaraie and Geesink 2006; Geesink et al. 2006; Koohmaraie 1992).
O’Halloran et al. (1997) studied the relationship between μ-calpain and meat ten-
derness and found out that muscle with lower μ-calpain activity at 3 h postmortem
had higher protein degradation level, which meant that muscle was tender with faster
autolysis of μ-calpain. At 7 d postmortem, the activity of m-calpain showed no
obvious change in all three groups, indicating that the differences in meat tenderness
in three groups were not induced by m-calpain.
8.2.4 The Degradation of μ-Calpain 80 kDa Subunit
The 80 kDa subunit of μ-calpain degraded slowly in all the three groups during
postmortem (Fig. 8.4). The degradation rate of μ-calpain in tender group was
significantly higher than the middle and tough groups at 1, 6, 12, and 72 h postmor-
tem (P < 0.05). Overall, the degradation rate of μ-calpain was: tender group > middle
group > tough group.
Studies have shown that the autolysis and activation of μ-calpain were related to
the degradation of myofibrillar proteins at early postmortem (Melody et al. 2004).
Most of the protein degradation and tenderization was induced by μ-calpain activa-
tion. μ-calpain with faster autolysis rate showed earlier activation and faster deacti-
vation (Killefer and Koohmaraie 1994). Kim et al. (2013) studied the autolysis of
μ-calpain in mutton during postmortem aging by western blotting and discovered
1.3
Tender group
a a a
1.2
Relative degradation rate
a Middle group
a a a
1.2 a ab Tough group
a ab b b b
ab b b
1.1 a
b c c c c
c b b
1.1 b c
b
b
1.0
1.0
0.9
30min 1h 2h 6h 12h 24h 48h 72h 5d 7d
Postmortem time
Fig. 8.4 Degradation rate of μ-calpain 80 kDa subunit (Du 2018)
that the autolysis and activation degree of μ-calpain was positively related to protein
degradation. Muscle with higher content of μ-calpain intact 80 kDa subunit
presented lower proteolysis degradation and lower tenderness (Cruzen et al. 2014).
In the present study, the degradation rate of μ-calpain in tender group was higher
than middle group, than that of in tough group, indicating that the activation degree
and activity of μ-calpain were the highest in tender group and the lowest in tough
group, which was consistent with the results of casein zymography. Muscle with
different tenderness had different phosphorylation level of sarcoplasmic proteins.
Tender muscle presented lower phosphorylation level of sarcoplasmic proteins but
higher activity of μ-calpain, which demonstrated that phosphorylation of sarcoplas-
mic proteins may have negatively regulated μ-calpain activity and finally influenced
meat tenderness during postmortem.
8.2.5 The Degradation of Calpastatin
During postmortem, the 130 kDa calpastatin degraded gradually in all three groups
(Fig. 8.5). The 130 kDa calpastatin in tender and middle groups entirely degraded at
6 h and 5 d postmortem, respectively. However, calpastatin could still be detected in
tough group at 7 d postmortem. Calpastatin in tender group degraded faster than
middle group, than that of in the tough group.
As one of the well-characterized parts in calpain system, the endogenous
calpastatin was the only known protein inhibitor specific for μ-calpain and
m-calpain (Crawford 1990). Each calpastatin could inhibit four calpain molecules
to limit postmortem muscle proteolysis and meat tenderization (Goll et al. 2003;
Kent et al. 2004). Although the degradation of calpastatin did not equal to the loss of
8.3 Effects of Phosphorylation of Sarcoplasmic Proteins on μ-Calpain Activity at. . . 155
Fig. 8.5 Degradation of calpastatin during postmortem (Du 2018)
inhibitory activity, the inhibitory activity of calpastatin was weakened by degrada-

tion (Huang et al. 2014). Following the degradation of calpastatin, the muscle
became tender during postmortem (Geesink et al. 2005; Boehm et al. 1998).
Tough meat showed higher level of calpastatin, because of the inhibitory ability of
calpastatin to the proteolysis activity of μ-calpain (Kemp et al. 2010). Tender group
had a faster degradation rate of calpastatin, indicating the faster deactivation rate of
calpastatin. Therefore, the inhibitory ability of calpastatin to μ-calpain was lowest in
tender group. Salamino et al. (1994) studied the phosphorylation of calpastatin and
found out that, compared to dephosphorylated calpastatin, phosphorylated
calpastatin showed greater inhibition to m-calpain. In the present study, tough
group had higher phosphorylation level of sarcoplasmic proteins and higher degra-
dation level of calpastatin, which demonstrated that phosphorylation might play
negative roles in meat tenderization by enhancing the inhibition of calpastatin to
μ-calpain activity and then inhibiting protein proteolysis at postmortem.

on μ-Calpain Activity at Different Incubation
Temperature
The indirectly effects of phosphorylation on the activity of μ-calpain and the

sensitivity of μ-calpain to temperature were investigated in this part. Sarcoplasmic
proteins were extracted from three Fat Tail Han sheep (8 months) at 30 min
postmortem, adjusted to 12 μg/μL and subjected to three treatments: (1) 25 U/
100 μg protein alkaline phosphatase (AP); (2) One tablet/10 mL supernatant phos-
phatase inhibitor (PI); (3) Control. For temperature sensitivity analysis, sarcoplasmic
protein was treated with AP and phosphatase inhibitor (PI) at 4, 25, and 37 C,
respectively.

Proteins
To modulate the phosphorylation level of proteins, prepared sarcoplasmic proteins

were treated with or without AP. As expected, incubation of proteins with AP
remarkably decreased the phosphorylation of sarcoplasmic proteins, since the inten-
sity of protein bands of the AP group on the Pro-Q Diamond stained gels was
obviously lower than that of the other two groups (Fig. 8.6a, c, g, h). The phosphor-
ylation level of sarcoplasmic proteins in three treatments at different temperatures
was evaluated by the ratio of phosphoproteins to total proteins (Fig. 8.6). The results
showed that the intensity of Pro-Q Diamond stained proteins in AP group was
significantly lower compared to other groups during the 72 h incubation at both
4 and 25 C (P < 0.05) (Fig. 8.6a, c, g, h). AP dephosphorylated proteins, while
phosphatase inhibitors inhibited protein dephosphorylation both in vivo and in vitro
(Sharma et al. 2014; Chen et al. 2016). These two chemicals were often used to
modulate protein phosphorylation status. At all incubation temperatures, PI group
showed a greater intensity in Pro-Q Diamond stained proteins and a higher phos-
phorylation level compared to control group. Overall, AP group had the lowest
intensity, indicating the lowest phosphorylation level of proteins, which meant that
both AP and phosphatase inhibitor worked in this study. The high, middle, and low
protein phosphorylation level of PI, control, and AP groups revealed that protein
phosphorylation status could be effectively regulated by AP and PI.
8.3.2 μ- and m-Calpain Activity
Generally, the 80 kDa subunit of μ-calpain degraded slowly in all three groups
during incubation (Fig. 8.7). Comparatively, the AP group showed a higher level and
a faster rate of μ-calpain degradation than the control and PI groups. As the
temperature increased, the degradation of μ-calpain increased due to the intensity
of degraded μ-calpain bands increased in all three groups. It has been reported that
earlier autolysis/degradation of μ-calpain was associated with earlier activation of
μ-calpain in postmortem muscle. The degradation degree of μ-calpain was positively
correlated with μ-calpain activity (Melody et al. 2004). Thus, the activity of
μ-calpain was supposed to be higher in AP group compared to other two groups.
At 4 C and 37 C, the 80 kDa subunit of μ-calpain in control group degraded
relatively faster than in PI group, indicating that the activity of μ-calpain in control
group was higher than in the PI group of high protein phosphorylation level.
However, no difference in the degradation of μ-calpain was detected between control
and PI samples incubated at 25 C. The optimal temperature for μ-calpain activity
was 25 C (Boehm et al. 1998). At this temperature, μ-calpain had the best prote-
olysis ability and maximal activity. In other words, the degradation rate of μ-calpain
was the fastest at this temperature. In this study, the AP was more effective than PI in
Fig. 8.6 Phosphorylation of sarcoplasmic proteins in three groups incubated at 4, 25, and 37 C. (a), (b) Samples incubated at 4 C. (c), (d) Samples incubated
8.3 Effects of Phosphorylation of Sarcoplasmic Proteins on μ-Calpain Activity at. . .
at 25 C. (e), (f) Samples incubated at 37 C. (a), (c), (e) SDS-PAGE gel stained by Pro-Q Diamond stains. (b), (d), (f) SDS-PAGE gel stained by Ruby stains.
(g), (h), (i) Relative phosphorylation level of sarcoplasmic proteins in three treatments incubated at 4, 25, and 37 C, respectively. Values with different letters
show significant difference in the results in different groups at the same incubation time (P < 0.05). AP Alkaline phosphatase, PI Phosphatase inhibitor (Du et al.
2017a)
157
AP Control PI AP Control PI
St 0.5h 2h 6h 12h 0.5h 2h 6h 12h 0.5h 2h 6h 12h St 24h 48h 72h 24h 48h 72h 24h 48h 72h
Fig. 8.7 Western blotting analysis during incubation at 4, 25, and 37 C. (a), (b), (c) The
degradation of μ-calpain 80 kDa subunit in three groups incubated at 4, 25, and 37 C, respectively.
AP Alkaline phosphatase, PI Phosphatase inhibitor (Du et al. 2017a)
regulating protein phosphorylation status, because the difference in protein phos-

phorylation levels between AP and control samples was greater than that between
control and PI groups (Fig. 8.6g, h, i). Consequently, the difference in the degrada-
tion of 80 kDa μ-calpain subunit between AP sample and control was greater than
that between control and PI groups when incubated at 4 C and 37 C. The
degradation of 80 kDa subunit showed no difference between control and PI samples
when incubated at 25 C. This could be the optimal temperature and thus the high
activity of μ-calpain eliminated the effect of the minute difference in protein
phosphorylation on the autolysis of μ-calpain (Boehm et al. 1998).
Phosphorylation altered the physiological functions of proteins by changing their
structure or biological activities. Phosphorylation of sarcoplasmic proteins in
response to electrical stimulation increased the activity of metabolic enzymes
(Li et al. 2015). The phosphorylation of μ-calpain catalyzed by extracellular protein
kinases (ERK) not only enhanced its activity, but also promoted its secretion (Xu and
Deng 2004). In contrast, the data obtained in this study showed that phosphorylation
may negatively regulate μ-calpain degradation and activation. In addition, the
influence of phosphorylation on μ-calpain degradation was greater at 37 C, which
reduced at 25 C, indicating that temperature could influence the sensitivity of
μ-calpain to phosphorylation.
In the detection of μ-calpain and m-calpain by casein zymography in the present
study, casein gels were incubated in the presence of 4 mM Ca2+. This concentration
of Ca2+ can activate both μ-calpain and m-calpain to degrade casein at their locations
in the gel. Thus, the content of detected μ-calpain remained 80 or 78 kDa subunit
St 0.5h 2h 6h 12h 0.5h 2h 6h 12h 0.5h 2h 6h 12h St 24h 48h72h 24h 48h 72h 24h 48h 72h
A µ-calpain
m-calpain
B
µ-calpain
m-calpain
C µ-calpain
m-calpain
Fig. 8.8 Casein zymography gel analysis during incubation at 4, 25, and 37 C. (a), (b), (c) μ- and
m-calpain in three groups incubated at 4, 25, and 37 C, respectively. AP Alkaline phosphatase; PI
Phosphatase inhibitor (Du et al. 2017a)
which kept its proteolysis ability. The results showed that the autolyzed μ-calpain
was only detected in AP samples incubated at 37 C for 48 h and in both control and
PI samples incubated at 37 C for 72 h (Fig. 8.8c). After 48 h incubation at 37 C, the
content of autolyzed μ-calpain increased more enough to be activated in gels to
hydrolyze casein. The less unautolyzed μ-calpain in AP samples incubated at any
temperature in this study (Fig. 8.8) demonstrated that the autolysis degree of
μ-calpain in this group was greater than that of the other two groups, which was
consistent with the results of western blotting analysis of μ-calpain degradation.
The m-calpain, same as μ-calpain, consisted of an 80 kDa catalytic subunit and a
28 kDa regulatory subunit. m-calpain was also calcium dependent and could be
activated by autolysis in the presence of calcium (Cong et al. 1989). In most cases,
the concentration of Ca2+ in postmortem muscle was too low to activate m-calpain,
thus, m-calpain may not be crucial in meat tenderization postmortem (Koohmaraie
and Geesink 2006). However, autolyzed m-calpain was detected in pork stored in
refrigerator for 6 days (Pomponio et al. 2008). Pomponio and Ertbjerg (2012)
reported that m-calpain was activated by the high temperature in pork. In the present
study, noticeable autolysis of m-calpain happened only in AP and control samples
incubated at 37 C for 72 h, indicating that high temperature activated m-calpain.
Meanwhile, the content of unautolyzed m-calpain was much higher in higher
phosphorylated sarcoplasmic proteins. m-calpain was phosphorylated by PKA or
ERK. Phosphorylation of m-calpain increased its activity (Xu and Deng 2004; Xu
and Deng 2006a, b; Leloup et al. 2010). Some literature reported that phosphoryla-
tion of m-calpain at serine 369 kept it in an inactive state (Shiraha et al. 2002). In our
study, the autolysis degree of m-calpain was greater in AP samples than in the other
two groups, showing that phosphorylation inhibited m-calpain activation in vitro.
8.4 Effects of Phosphorylation on μ-Calpain Activity

at Different Incubation Temperature In Vitro
The effects of AP dephosphorylation and PKA phosphorylation on μ-calpain activity

and sensitivity to temperature were investigated in this part. The purified μ-calpain
was treated with AP or PKA for 30 min at 30 C to regulate its phosphorylation level.
Samples were then incubated at controlled freezing point (1), 4, 25, and 37 C,
respectively. The results showed that PKA and AP had no influence on pH values of
incubation solution. At 1 and 4 C, the maximum degradation rate of μ-calpain was
detected in AP group and the minimum was in control group. Low temperature of
controlled freezing point prevented dephosphorylation and phosphorylation progres-
sion and delayed μ-calpain degradation. Higher incubation temperature of 4, 25, and
37 C increased μ-calpain degradation. Two degradation products with about 50 kDa
from μ-calpain were identified, of which the intensity was also lower in control
group than in the other two groups. These observations demonstrated that AP
dephosphorylation and PKA phosphorylation of μ-calpain promoted μ-calpain autol-
ysis and activation.
8.4.1 The pH Values of μ-Calpain Solution Measured Before

and After Treated with AP/PKA
The pH values were presented in Table 8.1. The results showed that there was no
difference in pH values among the three treatments either before or after incubation
(P > 0.05). pH was an important factor affecting μ-calpain activity in postmortem
muscle (Koohmaraie et al. 1986). It has been reported that μ-calpain was optimally
active at pH 7.5 and autolyzed rapidly in high pH muscle in which myofibrillar
proteins degraded fast (Lomiwes et al. 2014a; Pulford et al. 2009). In the present
study, no difference in pH values indicated that the effect of pH on μ-calpain activity
was not significantly different between the three treatments.
Table 8.1 The pH values of μ-calpain solution measured before and after incubation at 30 C for
30 min (Du et al. 2017b)
Treatment 0 min 30 min
AP 6.72 0.06aA 6.59 0.02aB
Control 6.77 0.01aA 6.60 0.02aB
PKA 6.74 0.06aA 6.63 0.02aB
a values with the same lowercase within each column do not differ (P > 0.05), AB within the same
row, values with different uppercase letters differ (P < 0.05)
8.4 Effects of Phosphorylation on μ-Calpain Activity at Different Incubation. . . 161
8.4.2 The Phosphorylation Level of μ-Calpain
To estimate the effect of PKA and AP on μ-calpain phosphorylation, the phosphor-

ylation level of μ-calpain was measured after μ-calpain was incubated with or
without PKA/AP. Figure 8.9a, b showed the phosphorylated and total μ-calpain,
respectively. The intensity of phosphorylated μ-calpain was the highest in PKA
group and the lowest in AP group, while there was no significant difference in the
intensity of total μ-calpain among the three treatments. The phosphorylation level of
μ-calpain was PKA group > control group > AP group (Fig. 8.9c). This result
2.6 a
2.4
2.2
2.0
1.8
1.6
b
1.4
1.2 c
1.0
0.8
AP C PKA
Fig. 8.9 Phosphorylation level of μ-calpain after incubation with AP and PKA at 30 C for 30 min.
(a–b), SDS-PAGE gels stained with Pro-Q Diamond (a) and SYPRO Ruby (b). (c) Relative
phosphorylation level of μ-calpain. Values with different letters are significantly different
(P < 0.05). AP Alkaline phosphatase, PKA Protein kinase A (Du et al. 2017b)
2.0 2.5 a a
Relative phosphorylation
2.0 a a
1.5 ab
AP 1.5 bb bb bb b AP
1.0
Control 1.0 Control
level
level
0.5 PKA PKA
0.5
0.0 0.0
10 20 40 60 10 20 40 60
Incubation time/min Incubation time/min
2.0 3.0 a
a
2.5 a
1.5 b baba b
b aba AP 2.0
ab AP
b
1.0 Control 1.5 b b Control
level
level
PKA 1.0 PKA

0.5
0.5
0.0 0.0
10 20 40 60 10 20 40 60
Fig. 8.10 Phosphorylation level of μ-calpain incubated at different temperatures and in the
presence or absence of PKA/AP. (a–d) Relative phosphorylation level of μ-calpain incubated at
controlled freezing point (a), 4 C (b), 25 C (c), and 37 C (d). ab At the same time point, values
lacking a common letter are significantly different (P < 0.05). AP Alkaline phosphatase, PKA
Protein kinase A (Du et al. 2017b)
demonstrated that, as expected, PKA and AP had effectively phosphorylated and

dephosphorylated μ-calpain after incubation at 30 C for 30 min, respectively.
The incubation of μ-calpain at controlled freezing point (1 C), 4, 25, and 37 C
for 10, 20, 40, and 60 min was to study the impact of temperature on μ-calpain
phosphorylation and dephosphorylation. Generally, for incubation temperature of
4, 25, and 37 C, the phosphorylation level of μ-calpain in control group was higher
than in AP group, but lower than in PKA group (Fig. 8.10b, c, d), which indicated
that phosphorylation and dephosphorylation process effectively kept three treat-
ments maintain established phosphorylation state. However, there was no significant
difference in phosphorylation level of μ-calpain among the three treatments when
incubated at controlled freezing point (P > 0.05) (Fig. 8.10a). Food products stored
at controlled freezing point (also called superchilling storage), between freezing and
refrigeration where ice crystals could not be generated (Beaufort et al. 2009), have
been an increasingly popular way to maintain food freshness and high quality and
increase their shelf life (Kaale et al. 2011). Under such storage condition, the
microbial growth and lipid oxidation in surimi were reduced, and the proteolytic
degradation was limited (Liu et al. 2014). Previous studies of our group about the
effects of controlled freezing point storage on the protein phosphorylation level of
mutton showed that controlled freezing point reduced protein phosphorylation level
at early postmortem by inhibiting the activity of protein kinases (Zhang et al. 2016;
Li et al. 2016), which was consistent with this research. Compared with refrigeration,
controlled freezing point could delay muscle aging (Li et al. 2016). Therefore, the
limited biochemistry activities under superchilling resulted in insignificant
8.4 Effects of Phosphorylation on μ-Calpain Activity at Different Incubation. . . 163
δAε
δBε
δCε
δDε
2.0
Ca Ca Da Da
δEε
ε 1.5 Ba Db
Aa Cb
AbAb BbBb
1.0 AP
Control
0.5 PKA
0.0
10 20 40 60
Incubation time/min
2.0
Ca Ca
Ba Cab Dab
Cb
1.5 Bab Cb
δFε
ε Aa Ab
Ac
Bb
AP
1.0
Control
PKA
0.5
0.0
10 20 40 60
Incubation time/min
1.6 A A A A
A A
1.4
δGε
ε 1.2 BB B
1.0 C AP
0.8 C Control
0.6 C PKA
0.4
0.2
0.0
10 20 40 60
Incubatiom time/min
Fig. 8.11 Degradation of 80 kDa μ-calpain subunit during incubation at different temperatures. (a–
d) The degradation of 80 kDa μ-calpain subunit incubated at controlled freezing point (a), 4 (b),
25 (c), 37 C (d). (e–g) Relative degradation rate of 80 kDa μ-calpain subunit incubated at
controlled freezing point (e), 4 (f), 25 C (g). ab At the same time point, values lacking a common
difference in phosphorylation level of μ-calpain when incubating at controlled

freezing point.
8.4.3 The Degradation of μ-Calpain
The detection of μ-calpain degradation has previously been used to evaluate

μ-calpain activity and to analyze the existing state of μ-calpain in real time (Lomiwes
et al. 2014b; Melody et al. 2004). The μ-calpain degraded from 80 kDa to 76 kDa
peptide in all three groups during incubation (Fig. 8.11). A gradual increase of
76 kDa μ-calpain was observed at controlled freezing point and 4 C (Fig. 8.11a, b).
With the increase of incubation temperature, μ-calpain degraded faster. After incu-
bation at 37 C for 10 min, it was no longer possible to detect any 78 or 76 kDa
subunit of μ-calpain in all three groups (Fig. 8.11d). At controlled freezing point and
4 C, μ-calpain in AP group had the faster degradation rate than in the other two
groups. Meanwhile, the degradation rate of μ-calpain in PKA group was higher than
that in control group (Fig. 8.11e, f). However, there was no significant difference in
the degradation rate of μ-calpain among AP, control, and PKA groups at 25 C
(P > 0.05) (Fig. 8.11g). Generally, the 80 kDa subunit of μ-calpain was degraded to
a 76 kDa form through a 78 kDa intermediate by autolysis for its activation. A faster
degradation rate of μ-calpain suggested a higher activity of this enzyme (Lomiwes
et al. 2014a). Therefore, the higher degradation rate of μ-calpain in AP group at
controlled freezing point and 4 C revealed that dephosphorylation contributed to
higher activity of μ-calpain. This was in agreement with our previous study which
showed an increase in μ-calpain activity when sarcoplasmic proteins were
dephosphorylated by AP (Du et al. 2017a). PKC phosphorylation of both μ- and
m-calpain activated these enzymes, leading to enhanced invasion and migration of
cancer cells (Xu and Deng 2006a). MAPK (mitogen-activated protein kinase)/ERK1
and MAPK/ERK2 directly phosphorylated μ- and m-calpain in vitro to promote their
activation, secretion, and proteolysis ability (Xu and Deng 2004). However, activa-
tion of m-calpain was limited by PKA phosphorylation (Shiraha et al. 2002). The
opposite effects of PKA and ERK phosphorylation on m-calpain activity demon-
strated that different types of protein kinase may have different regulatory roles on
calpain. However, the effect of PKA phosphorylation on μ-calpain activity still
remains unknown. The present study showed that phosphorylation of μ-calpain by
PKA led to increased degradation of μ-calpain compared to control group, indicating
that PKA phosphorylation increased μ-calpain activity.
Controlled freezing point storage has been an effective approach to extend meat
shelf life and maintain meat quality by reducing microbial growth and limiting
Fig. 8.11 (continued) letter are significantly different (P < 0.05). ABCD Within the same group,
values lacking a common letter are significantly different (P < 0.05). AP Alkaline phosphatase,
PKA Protein kinase A (Du et al. 2017b)
biochemical reactions in meat (Kaale et al. 2011; Liu et al. 2014). The controlled
enzyme activity and limited proteolytic degradation delayed the aging of meat. In the
present study, no difference in the degradation rate of 80 kDa μ-calpain subunit was
detected between control and PKA groups after incubated at controlled freezing
point for 10 and 20 min (Fig. 8.11e). Considering the reduced phosphorylation level
of μ-calpain in PKA group at controlled freezing point, the results indicated that
controlled freezing point incubation decreased μ-calpain activity by delaying its
phosphorylation process. μ-calpain was a temperature sensitive enzyme. Higher
incubation temperature rapidly activated μ-calpain followed by an increase in the
degradation of μ-calpain (Pomponio and Ertbjerg 2012). In addition, the activity of
μ-calpain was optimal at 25 C (Pomponio and Ertbjerg 2012), resulting in the
eliminated difference among the three groups. After incubation at 25 C for 20 min
or at 37 C for 10 min, the intensity of both intact and autolyzed μ-calpain decreased,
suggesting that the 76 kDa peptide of μ-calpain may further degrade to smaller
forms.
The further degradation products of μ-calpain catalytic subunit have never been
studied in previous research. Two specific bands at about 50 kDa were detected on
SDS-PAGE gels by western blotting (Fig. 8.12a, b, c, d), which were probably the
further degradation products of 76 kDa μ-calpain peptide. To prove it, proteins in
these two bands were identified by LC-MS/MS. As shown in Table 8.2, μ-calpain
was identified in both bands, proving that the 76 kDa μ-calpain peptide degraded to
about 50 kDa peptide after incubation. The intensity of band 1 and band 2 increased
during incubation at controlled freezing point and 4 C (Fig. 8.12a, b) and was
higher in AP and PKA groups than in control group. When incubation temperature
increased to 25 C, band 1 disappeared after incubation for 10 min and the intensity
of band 2 decreased after incubation for 40 min (Fig. 8.12c). Despite the degradation
rate of μ-calpain from 80 kDa to 76 kDa showed no significant difference among the
three groups, the content of about 50 kDa products was lower in control group than
in the other two groups when incubated at 25 C (Fig. 8.12c), indicating that the
degradation rate of μ-calpain was faster in AP and PKA groups than in control group.
At 37 C, it was no longer possible to detect band 1 and the intensity of band
2 significantly decreased (Fig. 8.12d, h), which meant that about 50 kDa peptide of
μ-calpain may degrade to even smaller molecules. All these results showed that
degradation of μ-calpain was phosphorylation and temperature dependent. The AP
dephosphorylation and PKA phosphorylation of μ-calpain promoted its degradation
and activation.

on μ-Calpain Activity at Different Ca2+ Concentrations
The indirect effects of phosphorylation on the activity of μ-calpain and the sensitiv-
ity of μ-calpain to Ca2+ were investigated in this part. Sarcoplasmic proteins were
extracted from three Fat Tail Han sheep (8 months) at 30 min postmortem, adjusted
δAε
δBε
δCε
ε
δDε
ε
2.5 0.8
Eε δFε
ε B
Ba 0.7
Relative degradation
2.0 B
0.6
AP 0.5 AP
1.5
amount
Ba Control
amount
B Control
0.4 Aa Aa Aa PKA
1.0 PKA
Aab Aa Aa Aa 0.3
Aa Aa Bb Aa Aa
0.5 0.2 Ab
Ab Ab Ab Ab
Ab 0.1 Ab
0.0 0.0
10 20 40 60 10 20 40 60
3.5 0.3
G A
3.0
B δHε
ε 0.3
A
A
2.5 C
AP 0.2 AP
B Control Control
amount
amount
2.0 BC
PKA 0.2 PKA
1.5 Aa A A B B
AB A
1.0 0.1 BB
AbAb
0.1 Ba Ba
0.5 CbCb CbCb
0.0 0.0
10 20 40 60 10 20 40 60
Fig. 8.12 Degradation of 80 kDa μ-calpain subunit to about 50 kDa peptides. (a–d) Detection of
about 50 kDa products after incubation of μ-calpain at controlled freezing point (a), 4 (b), 25 (c),
37 C (d). (e–h) Relative intensity of μ-calpain smaller peptides in three groups incubated at
controlled freezing point (e), 4 (f), 25 (g), 37 C (h). ab At the same time point, values lacking a
common letter are significantly different (P < 0.05). ABC Within the same group, values lacking a
common letter are significantly different (P < 0.05). AP Alkaline phosphatase, PKA Protein kinase
A (Du et al. 2017b)
Table 8.2 LC-MS/MS identified proteins in about 50 kDa protein bands on SDS-PAGE gels
(Du et al. 2017b)
Band Accession Protein name Score Mass Matches Sequences
1 Q27970 Calpain-1 catalytic subunit 2411 82,782 137 (91) 25 (24)
2 Q27970 Calpain-1 catalytic subunit 6291 82,782 325 (235) 26 (24)
to 12 μg/μL, and subjected to three treatments: (1) 25 U/100 μg protein AP (AP);

(2) One tablet/10 mL supernatant phosphatase inhibitor (PI); (3) Control. For
calcium sensitivity analysis, samples treated with AP and PI were incubated at
0.01, 0.05, 0.1, and 1 mM Ca2+, respectively.

Proteins
The phosphorylation level of prepared sarcoplasmic proteins was measured to verify

whether AP and PI worked effectively in regulating protein phosphorylation. Results
showed that the phosphorylation level of PI group was significantly higher than
control, which was significantly higher than AP group (P < 0.05) (Fig. 8.13).
8.5.2 μ-Calpain Activity
The degradation of μ-calpain and the corresponding degradation rate in the presence
of Ca2+ were shown in Fig. 8.14. In the presence of 0.01, 0.05, and 0.1 mM Ca2+, the
degradation of 80 kDa μ-calpain subunit to 76 kDa form was fastest in AP samples,
of which the rate was significantly higher than that of the other two groups
(P < 0.05). PI samples had the lowest degradation rate (Fig. 8.14a, b, c). The
80 kDa subunit of μ-calpain remarkably degraded when the Ca2+ concentration
was higher than 0.05 mM, but the degradation level remained unchanged when
[Ca2+] ¼ 0.1 mM. The differences in degradation rate among the three groups
disappeared when Ca2+ concentration was 1 mM. μ-calpain was a calcium dependent
protease and required micro-molar of calcium for its activation. The optimal Ca2+
concentration for its activity has been intensively studied (Biswas et al. 2016).
Injection of CaCl2 increased calpain activity and improved meat tenderness
(Jaturasitha et al. 2004). And injection of CaCl2 also activated μ-calpain, which
eventually resulted in activity loss due to autolysis (Koohmaraie et al. 1989;
Koohmaraie et al. 1990). The increased Ca2+ activated μ-calpain and promoted
the degradation of μ-calpain at the same time. The AP group had a greater degrada-
tion degree of μ-calpain at 0.01, 0.05, and 0.1 mM Ca2+ concentrations, which
indicated that μ-calpain with lower phosphorylation had higher activity, which was
consistent with experiment 1. As the level of phosphorylation increased, Ca2+
（A）
0.7 a
b
0.6
c
0.5
0.4
0.3
AP C PI
（B）
Fig. 8.13 Phosphorylation of sarcoplasmic proteins before incubating at different Ca2+ concentra-
tion. (a) Left: SDS-PAGE gel stained by Pro-Q Diamond stains; right: SDS-PAGE gel stained by
Ruby stains. (b) Relative phosphorylation level of three treatments (Du et al. 2017a)
concentration required for μ-calpain activation increased. Furthermore, as the con-

centration of Ca2+ increased, the difference in μ-calpain degradation among AP,
control, and PI groups reduced. The effect of phosphorylation on μ-calpain activity
reduced and even disappeared as Ca2+ concentration increased. This could be
explained by the increased activity of μ-calpain in the presence of calcium.
Aε
ε
Bε
Cε
Dε
1.6 a
a
1.4 AP
a
δEε 1.2
rate
1.0 a
b b b
0.8 b
c c c
0.6 c
0.4
0 20 40 60
Incubation time/min
1.8
a AP
1.6
δF ε 1.4 a Control
1.2 a a
rate
1.0 b
b
0.8 b c
0.6 cb
0.4 c b
0 20 40 60
Incubation time/min
1.1 a a a a
1.0 b b a AP
b b
δG ε 0.9 c
0.8
rate
0.7 c c
0.6
0.5
0.4
0 20 40 60
Incubation time/min
1.6 AP
a
1.4 Control
a
ba b PI
δH ε 1.2
c
rate
1.0
0.8
0.6
0.4
0 20 40 60
Incubation time/min
Fig. 8.14 Western blotting analysis during incubation at different Ca2+ concentration. (a), (b), (c),
(d) The degradation of μ-calpain 80 kDa subunit in three groups incubated at 0.01, 0.05, 0.1, and
1 mM Ca2+, respectively. (e), (f), (g), (h) Relative degradation rate of μ-calpain 80 kDa subunit in
8.6 Effects of Phosphorylation on μ-Calpain Activity

at Different Ca2+ Concentrations In Vitro
The effect of phosphorylation/dephosphorylation regulated by PKA and AP on

μ-calpain activity at different Ca2+ concentrations was investigated in this part.
The purified μ-calpain was treated with AP or PKA at 0.01, 0.05, 0.1, and 1 mM
Ca2+. The pH value decreased in the AP group but remained stable in the control and
PKA groups during incubation. Except for samples incubated at 0.01 and 0.1 mM
Ca2+ for more than 20 min, μ-calpain incubated with PKA showed a higher level of
autolysis than control, but lower than the AP group. The content of α-helix structure
of μ-calpain increased as phosphorylation level rose. Phosphorylation of μ-calpain at
serines 255, 256, 476, 417, and 420 was identified. PKA catalyzed μ-calpain
phosphorylation at serines 255, 256, and 476, located at domains II and III, posi-
tively regulated μ-calpain activity. These data demonstrated that dephosphorylation
and PKA phosphorylation positively regulated μ-calpain activity, which was limited
by increased Ca2+ concentration.
8.6.1 The Changes in pH Values During Incubation
The pH values of μ-calpain solutions treated with or without AP/PKA were

presented in Fig. 8.15. Before incubation with AP and PKA, there was no significant
difference in the pH values of μ-calpain solutions. After 30 min incubation, the pH of
AP group decreased from 6.91 to 6.70, which was significantly lower than that of the
other two groups (P < 0.05). There was no difference in pH values between the PKA
group and the control (P > 0.05). AP was a hydrolase that catalyzed the protein
dephosphorylation. The phosphate group removed from μ-calpain by AP was the
acidic ingredient in AP group that reduced the pH value (Longo et al. 2015).
8.6.2 The Phosphorylation of μ-Calpain
The phosphorylation level of μ-calpain after incubation with AP and PKA is shown
in Fig. 8.16a, b, c. On the Pro-Q Diamond stain gels, the intensity of phosphorylated
μ-calpain band was the highest in PKA group and the lowest in AP group
(Fig. 8.16a). Correspondingly, μ-calpain in PKA group had the highest phosphory-
lation level, but the AP group had the lowest phosphorylation level (P < 0.05)
(Fig. 8.16c). The process of removing phosphate group from proteins induced by AP
Fig. 8.14 (continued) three groups incubated at 0.01, 0.05, 0.1, and 1 mM Ca2+, respectively. AP:
Alkaline phosphatase; PI: Phosphatase inhibitor (Du et al. 2017a)
8.6 Effects of Phosphorylation on μ-Calpain Activity at Different Ca2+. . . 171
8.0
a a a
7.0
A Bb a Bb Bb a a
6.0
5.0
AP
pH
4.0 Control
3.0 PKA
2.0
1.0
0.0
0 30 40 90
Incubation time/min
Fig. 8.15 The changes in pH values during incubation at 0, 30, 40, 90 min. Values with different
lowercase letters show significant difference in the results of different groups at the same incubation
time (P < 0.05). Values with different capital letters show significant difference in the results in
same groups at different incubation times (P < 0.05). AP Alkaline phosphatase; PKA Protein kinase
A (Du et al. 2018)
was called dephosphorylation. Conversely, PKA had the ability to catalyze proteins
phosphorylation at serine/threonine residues (Shiraha et al. 2002). These data
showed that AP and protein kinase A worked as expected in our experiment to
dephosphorylate and phosphorylate μ-calpain, respectively. μ-calpain with different
phosphorylation level was obtained after incubation with or without AP/PKA.
The phosphorylation level of μ-calpain in incubation with Ca2+ was also quanti-
fied. Generally, the phosphorylation of μ-calpain treated with PKA was greater than
that of control, which was greater than that of μ-calpain incubated with AP
(Fig. 8.16d, e, f, g). However, samples incubated in the presence of 0.05 and
0.1 mM Ca2+ showed no significant difference in phosphorylation level after incu-
bation for 20 min (Fig. 8.16f, g). As the Ca2+ concentration increased, an increase in
μ-calpain degradation level in all treatments was observed (Fig. 8.16a, b, c, d). The
content of intact and autolyzed μ-calpain differed between three groups and was
significantly different from the initial content of μ-calpain, leading to a variable and
undifferentiated phosphorylation level of μ-calpain in three treatments at later stage
of incubation. Overall, incubation with PKA or AP in the presence of ATP effec-
tively changed the phosphorylation state of μ-calpain.
8.6.3 The Degradation of μ-Calpain
To investigate the changes in μ-calpain activity induced by phosphorylation, the

degradation/autolysis of μ-calpain was evaluated by western blotting. μ-calpain
（A） 1.8
（C）
phosphorylation level
a
1.6
Relative
1.4 b
c
1.2
（B） 1.0
0.8
AP Control PKA
1.8 1.8
（D）（E）
a a
1.6 a 1.6
a a
bb
Relative
1.4 1.4
Relative
b b AP b AP
c c
1.2 Control 1.2 Control
PKA PKA
1.0 1.0
0.8 0.8
10 20 40 60 10 20 40 60
1.6 a 2.0 a
（F）（G）
ab 1.8 a a
1.4 b b
1.6 b
Relative
c AP
Relative
1.2 AP 1.4
Control Control
1.2 PKA
1.0 PKA
1.0
0.8 0.8
10 20 40 60 10 20 40 60
Fig. 8.16 Phosphorylation of μ-calpain in three groups after phosphorylation and dephosphoryla-
tion treatments and during incubation at different Ca2+ concentrations. (a), (b) SDS-PAGE
gels stained by Pro-Q Diamond stains and Ruby stains, respectively. (c) Relative phosphorylation
level of μ-calpain after phosphorylation and dephosphorylation treatments. (d), (e), (f), (g) Relative
phosphorylation level of μ-calpain in three treatments incubated at 0.01, 0.03, 0.05, 0.1 mM Ca2+,
respectively. Values with different letters show significant difference in the results in different
groups at the same incubation time (P < 0.05). AP Alkaline phosphatase, PKA Protein kinase A
(Du et al. 2018)
degraded gradually at all tested Ca2+ concentrations during incubation (Fig. 8.17a, b,
c, d). Increased degradation of μ-calpain was detected as Ca2+ concentration
increased. The degradation degree of AP treated μ-calpain was significantly higher
than that of control (P < 0.05), except for incubation at 0.1 mM Ca2+ after 20 min.
μ-calpain of PKA treatment showed reduced degradation when compared to AP
treated μ-calpain, but a higher degradation level than control except for samples
incubated at 0.01 and 0.1 mM Ca2+ for more than 20 min (Fig. 8.17e, f, g, h). The
activation of μ-calpain was paralleled by its autolysis of the intact 80 kDa large
subunit to the 78 kDa than 76 kDa form and was associated with proteolytic activity
of μ-calpain in postmortem muscles (Goll et al. 2003; Lomiwes et al. 2014a). A
previous study by our team discovered that protein phosphorylation level of tender
meat was lower than that of tough meat (Chen et al. 2016). Degradation of AP
treated myofibrillar proteins by μ-calpain was greater than untreated proteins
(Li et al. 2017). These studies indicated that dephosphorylation promotes proteolysis
Aε
Bε
Cε
Dε
5.0 a 8.0 a
δEε δFε a b
4.0 6.0
a b
3.0 c
rate
rate
a b bb AP 4.0 a AP
2.0 a ba b b c
cb Control a Control
bab c
1.0 PKA 2.0 PKA
0.0 0.0
10 20 40 60 10 20 40 60
8.0 a 4.0
δGε a a δHε a a
6.0 a a a b 3.0 a
b b b
rate
AP AP
rate
4.0 a a b 2.0 c
b Control Control
2.0 PKA 1.0 PKA
0.0 0.0
10 20 40 60 10 20 40 60
Fig. 8.17 Western blotting analysis during incubation at 0.01, 0.03, 0.05, and 0.1 mM Ca2+. (a),
(b), (c), (d) The degradation of μ-calpain 80 kDa subunit in three groups incubated at 0.01, 0.03,
0.05, and 0.1 mM Ca2+, respectively. (e), (f), (g), (h) Relative degradation rate of μ-calpain 80 kDa
subunit in three groups incubated at 0.01, 0.03, 0.05, and 0.1 mM Ca2+, respectively. Values with
different letters show significant difference in the results in different groups at the same incubation
time (P < 0.05). AP Alkaline phosphatase, PKA Protein kinase A (Du et al. 2018)
and plays a positive role in regulating μ-calpain activity. Further study revealed that
higher μ-calpain activity of sarcoplasmic proteins could be dephosphorylated by AP
(Du et al. 2017a). The activity of μ-calpain was maximum at pH near neutral (Goll
et al. 2003; Koohmaraie and Geesink 2006). Based on the determined pH values
presented in Fig. 8.15, PKA and control groups with a higher pH value should have a
higher μ-calpain activity than AP group. In other words, the degradation degree of
μ-calpain in PKA and control group was supposed to be higher than AP group under
such the pH condition without considering other factors. However, the degradation
degree of μ-calpain in AP group was significantly higher than that of PKA and
control groups, which indicated that dephosphorylation of μ-calpain promoted its
degradation and enhanced the activity of μ-calpain. The effects of dephosphorylation
on μ-calpain activity were greater than that of pH.
μ-calpain of PKA treatment degraded faster compared to control, which meant
that incubation of μ-calpain with PKA promoted its autolysis and activation.
μ-calpain can be phosphorylated by protein kinase like PKA, PKC, and extracellular
signal-regulated kinase (ERK) (Storr et al. 2011). Phosphorylation of μ-calpain and
m-calpain by MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-
regulated kinase) could lead to its activation as well as secretion, increasing migra-
tion and invasion of lung cancer cells (Xu and Deng 2004). The same result was
found by investigating the phosphorylation of μ-calpain and m-calpain catalyzed by
PKC (Xu and Deng 2006a). ERK directly phosphorylated m-calpain at serine 50 in
domain I, which was autolyzed when calpain was activated by calcium (Glading
et al. 2004). However, activation of calpain by epidermal growth factor (EGF) was
inhibited by PKA. PKA phosphorylated m-calpain at serine 369 in domain III, which
contained the characteristic C2 domains and was involved in structural changes
during calcium binding (Hanna et al. 2008; Moldoveanu et al. 2008). Phosphoryla-
tion of serine 369 could restrict domain movement and keep m-calpain in an inactive
state (Shiraha et al. 2002). All these studies demonstrated that phosphorylation of
calpain at different sites by different kinases had different effects on its activity.
Different from m-calpain, the present study showed that phosphorylation of
μ-calpain by PKA improved its degradation and led to activation. AP catalyzed the
removal of phosphate group from proteins to decrease protein phosphorylation level.
Its broad-spectrum dephosphorylation ability could limit phosphorylation of protein
by almost all kinds of protein kinases. In the present study, the increased autolysis of
μ-calpain after incubation with AP demonstrated that dephosphorylation of
μ-calpain by AP promoted its activation. In addition, the disparity of degradation
degree among the three treatments decreased when Ca2+ concentration increased.
After incubation at 0.1 mM Ca2+ for 20 min, the difference in degradation rate of
μ-calpain disappeared among three groups (P > 0.05), demonstrating that high
concentrations of Ca2+ eliminated the impact of phosphorylation/dephosphorylation
on μ-calpain activity. Further work is required to determine the influences of
phosphorylation and dephosphorylation on μ-calpain catalyzed by other protein
kinase and phosphatase.
8.6.4 Changes in μ-Calpain Secondary Structure
To determine the changes in secondary structures that responded to phosphorylation

or dephosphorylation of μ-calpain, circular dichroism spectroscopy was used to
characterize the secondary structures of μ-calpain treated with or without AP/PKA.
Table 8.3 Estimated secondary-structure contents of μ-calpain after phosphorylation and dephos-
phorylation treatments (Du et al. 2018)
Treatment α-helix β-sheet β-turn Random coil Total sum
AP 0.188 0.205 0.312 0.295 1
Control 0.591 0.409 0 0 1
PKA 0.799 0.001 0 0.199 1
The content of α-helix, β-sheet, β-turn, and random coil structures was presented in
Table 8.3. For all the three treatments, the sum of structure fractions of μ-calpain was
1, showing that the analysis met the requirements. Studies about μ-calpain structure
concentrated on the crystal structure, while circular dichroism has been rarely used.
α-helix and β-sheet were the essential second structures of μ-calpain (Edmunds et al.
1991). The circular dichroism spectra of μ-calpain showed a notable negative peak at
around 223 nm and a weaker negative peak at around 236 nm (Fig. 8.18). The
negative peak at around 223 nm, which represented α-helix structure (Edmunds et al.
1991), became larger as phosphorylation level of μ-calpain increased, indicating the
increased content of α-helix. This suggested that dephosphorylation of μ-calpain by
AP caused α-helical structure to break down, while PKA induced the formation of
α-helix. All the four domains of 80 kDa μ-calpain subunit contained α-helix,
however, α-helix mainly concentrated on domain II and domain IV, which also
contained five EF hands (Goll et al. 2003; Strobl et al. 2000). Any changes in α-helix
in μ-calpain may influence the combining of calcium to EF hands, the proteolysis,
and activity of μ-calpain. In addition, the content of β-sheet structure in AP and PKA
treated μ-calpain was lower, but random coil structure was higher than in control.
The lower content of β-sheet structure and the higher content of random coil
structure may be the reason why the activity of μ-calpain in AP and PKA groups
was significantly higher than that of control (Fig. 8.17). β-sheet principally existed in
catalytic domain (II) and regulatory domain (III) which plays important roles in
substrate binding and catalysis (Reverter et al. 2001; Strobl et al. 2000). The
difference in β-sheet, β-turn, and random coil content of μ-calpain among the three
treatments indicated that phosphorylation and dephosphorylation may be involved in
regulating the activity of μ-calpain by changing its secondary structure, sheltering or
exposing the binding sites of substrate proteins or calcium on μ-calpain.
8.6.5 Detection of Phosphorylated Peptides

and Phosphorylated Sites of μ-Calpain
A total of three unique phosphopeptides with five phosphorylated sites of μ-calpain

was identified in the present study. For PKA treated μ-calpain, the three
phosphopeptides and the five phosphorylation sites (serine 476, 417, 420, 255, and
256) were all determined. For the control and AP treated μ-calpain, the three unique
phosphopeptides and only two phosphorylation sites (serine 417 and serine 420)
20
15
10
5
MilliDegree
AP
0
210 215 220 225 230 235 240 245 250 Control
-5 PKA
-10
-15
-20
Wavelength/nm
Fig. 8.18 The influence of phosphorylation on the circular dichroism spectra of μ-calpain after
phosphorylation and dephosphoryaltion treatments. AP Alkaline phosphatase, PKA Protein kinase
A (Du et al. 2018)
Table 8.4 Q Exactive LC-MS/MS for the detection of phosphorylated peptides of μ-calpain after
phosphorylation and dephosphorylation treatments (Du et al. 2018)
Treatment Phosphorylated peptides Phosphorylation sites IonScorea
Standard – – –
AP EsGcsFLLALMQK Ser 417, Ser 420 19
Control EsGcsFLLALMQK Ser 417, Ser 420 26
PKA ARsEQFINLR Ser 476 44
EsGcsFLLALMQK Ser 417, Ser 420 65
GSLLGcSIDIssILDmEAVTFK Ser 255, Ser 256 76
a
The Mascot score of the phosphorylated peptides
were figured out (Table 8.4). At the same time, no phosphopeptides and phosphor-
ylation sites were determined in untreated standard sample, which meant that the
phosphorylation of μ-calpain occurred during incubation. In the present study, only
phosphoserine sites were determined. This was consistent with literature that phos-
phorylation of μ-calpain appeared mostly at serine residues (Vazquez et al. 2008).
The results showed that the number of phosphopeptides and phosphorylation sites of
μ-calpain identified in PKA group was much higher than that of the other two
groups. Although serine 417 and 420 were identified in all the three treatments,
the IonScore of the peptides was different. IonScore represented the Mascot score of
the identified phosphorylated peptides. Higher score meant a higher content of the
peptide and a higher accuracy of the matched result. Therefore, the higher IonScore
of phosphorylated peptides in PKA group than in control, which was higher than in
AP group, indicated that PKA effectively phosphorylated and AP dephosphorylated
8.7 The Inhibition of Calpastatin to the Activity of Phosphorylated μ-Calpain 177
μ-calpain at serine residues. This was in agreement with the measured μ-calpain
phosphorylation levels.
The serines 255 and 256 were in domain II and serines 417, 420, and 476 in
domain III of μ-calpain 80 kDa subunit. Phosphorylation of serines 255 and 256 may
change the ability of μ-calpain to catalyze substrate degradation. Regulatory domain
III was also involved in structural changes in μ-calpain. Thus, phosphorylation of
serine 476 may influence the self-regulatory activity of μ-calpain and facilitate
domain movement to activate μ-calpain (Shiraha et al. 2002). In addition, the
increased α-helix in domain II and IV of PKA treated μ-calpain suggested that
phosphorylation of μ-calpain at serines 255 and 256 regulated μ-calpain activity
by changing its structure (Shiraha et al. 2002; Storr et al. 2011). Phosphorylation of
serines in domain II and III could be the reason why μ-calpain incubated with PKA
had significantly higher activity.
8.6.6 The Ser-Phosphorylation Level of μ-Calpain
To confirm that PKA phosphorylated μ-calpain primarily at serine residues,

phosphoserine of μ-calpain was quantified by western blotting using antibodies
(Fig. 8.19). The band intensity of phosphorylated serine residues in PKA group
was obviously higher compared to control group, while AP group showed the lowest
content of phosphoserine at all Ca2+ concentrations. μ-calpain from bovine skeletal
muscle or human placenta has been reported to be phosphorylated at four serine
sites, three threonine sites, and two tyrosine sites (Vazquez et al. 2008). In the
present study, only phosphoserine was detected in μ-calpain. As PKA was a ser-
ine/threonine kinase, it was reasonable to detect no phosphotyrosine in the present
study. Nevertheless, it was unclear why the previously reported phosphothreonine
was not determined. The highest content of phosphoserine in PKA samples, but the
lowest in AP samples was consistent with the results of μ-calpain phosphorylation
level evaluated by SDS-PAGE and imaging analysis, indicating that PKA effectively
phosphorylated μ-calpain at serine residues.
8.7 The Inhibition of Calpastatin to the Activity

of Phosphorylated μ-Calpain
Calpastatin, as one of the well-characterized components in the calpain system, is the

only known protein inhibitor specific for μ-calpain and m-calpain. Each calpastatin
can inhibit four calpain molecules to limit postmortem muscle proteolysis and meat
tenderization. The effect of phosphorylation on the sensitivity of μ-calpain to the
inhibition induced by calpastatin was investigated in this part. Purified μ-calpain was
incubated with AP or PKA to regulate the phosphorylation level of μ-calpain.
Fig. 8.19 Detection of μ-calpain phosphorylated on serine residues during incubation at 0.01, 0.03,
0.05, and 0.1 mM Ca2+. (a), (b), (c), (d) Phosphorylation of μ-calpain at serine residues in three
groups incubated at 0.01, 0.03, 0.05, and 0.1 mM Ca2+, respectively (Du et al. 2018)
Accurately 25, 50, 100, and 150 units of AP/PKA treated μ-calpain were mixed with
the same amount of heat stable proteins and incubated at 4 C. In calpastatin-free
system, AP and PKA treated μ-calpain had higher proteolytic activity compared to
control. Intact AP treated μ-calpain degraded fastest in the 50, 100, and 150 units -
μ-calpain incubation systems. However, the degradation rate of μ-calpain in control
and PKA groups was non-significant in 100 and 150 units μ-calpain systems. Our
results demonstrated that, compared to dephosphorylated and control μ-calpain,
calpastatin presented greater inhibiting effect on PKA phosphorylated μ-calpain.
This study further enhanced the understanding of mechanism of μ-calpain activity
regulated by phosphorylation.
8.7.1 The pH Values of μ-Calpain Solution Measured Before

and After Treated with AP/PKA
The pH values of μ-calpain solution before and after incubation with AP/PKA were
showed in Table 8.5. No significant difference was detected between AP, control,
and PKA treated μ-calpain at 0 and 30 min (P > 0.05). Biochemical changes in
postmortem muscles were pH dependent, especially the activity of enzymes.
μ-Calpain was a neutral protease with an optimum pH 7.5 (Pulford et al. 2009).
Muscles with higher pH values have been shown to have greater degradation degree
Table 8.5 The pH values of μ-calpain solution measured before and after incubation at 30 C for
30 min (Du et al. 2019)
Treatment 0 min 30 min
AP 6.65 0.02 6.57 0.02
Control 6.68 0.03 6.61 0.03
PKA 6.65 0.02 6.60 0.02
AP Alkaline phosphatase, PKA Protein kinase A
and activation rate of μ-calpain than muscles with lower pH values (Bee et al. 2007).
Thus, the results indicated that the activity of μ-calpain was not affected by pH in
three treatments.
8.7.2 Phosphorylation Level of Heat Stable Proteins

and μ-Calpain
As shown in Fig. 8.20a, μ-calpain treated with AP had lower band intensity than that
of control after stained with Pro-Q Diamond gel. PKA group had the most abundant
phosphorylated μ-calpain. Non-significant difference in total proteins was deter-
mined among the three treatments (Fig. 8.20b). Correspondingly, the phosphoryla-
tion level of μ-calpain was the highest in PKA group and the lowest in AP group
(P < 0.05) (Fig. 8.20c). These data showed that phosphorylation level of μ-calpain
incubated with AP and PKA was significantly regulated as expected.
To determine whether AP/PKA addition, which altered the phosphorylation level
of μ-calpain, changed the phosphorylation status of heat stable proteins during
incubation at 4 C, the phosphorylation level of heat stable proteins after incubation
with μ-calpain for 1, 2, 12, and 24 h was detected. Heat stable proteins incubated
with 25, 50, 100, and 150 units of μ-calpain presented non-significant difference in
phosphorylation level among three treatments (P > 0.05) (Fig. 8.21i, j, k, l). The
intensity of protein bands on both Pro-Q Diamond and SYPRO Ruby stain gel
showed no significant difference among AP, control, and PKA treatments
(Fig. 8.21a–h). The amount of ATP added to regulate the phosphorylation level of
μ-calpain can only be used to phosphorylate less than 200 μg protein. When the
AP/PKA treated μ-calpain was mixed with heat stable proteins, the protein content of
each mixture was more than 7000 μg. Under such circumstances, the added ATP was
not adequate to influence the phosphorylation status of heat stable proteins and the
amount of AP/PKA was also limited in each treatment, resulting in the lack of
significant difference in phosphorylation level of proteins among three treatments in
the four incubation systems, which achieved the effect we desired.
（A）（B）
（C） 0.8 a
0.7
0.6
b
0.5 c
0.4
0.3
0.2
0.1
0.0
AP Control PKA
Fig. 8.20 Phosphorylation level of μ-calpain after incubation with AP and PKA at 30 C for
30 min. (a) SDS-PAGE gels stained with Pro-Q Diamond. (b) SDS-PAGE gels stained with
SYPRO Ruby. (c) Relative phosphorylation level of μ-calpain. Values with different letters are
significantly different (P < 0.05). AP Alkaline phosphatase, PKA Protein kinase A (Du et al. 2019)
8.7.3 The Activity of μ-Calpain
The 80 kDa subunit of intact μ-calpain progressively degrades to 78 kDa and then
76 kDa forms postmortem, which is considered as the activation process of μ-calpain
(Goll et al. 2003). In the present study, the degradation degree of μ-calpain increased
as the amount of μ-calpain added to the system increased (Fig. 8.22a, b, c, d). When
heat stable proteins were incubated with 25 units of μ-calpain, there was no signif-
icant degradation rate of μ-calpain among AP, control, and PKA groups (P > 0.05)
(Fig. 8.22e). Overexpression of calpastatin reduced the degradation extent and
degradation rate of μ-calpain, indicating a decline of postmortem proteolysis (Kent
et al. 2004). Therefore, the non-significant difference in degradation rate of μ-calpain
among the three treatments was caused by a relative excess of calpastatin to 25 units -
μ-calpain. When the amount of μ-calpain in incubation was more than 50 units, the
degree of degradation of μ-calpain in AP group was the greatest and the
corresponding degradation rate of μ-calpain was significantly higher than the other
two groups (P < 0.05) (Fig. 8.22b–d, f–h). μ-Calpain in AP and PKA groups had
significantly higher degradation rate than control group in calpastatin-free control
system during incubation (P < 0.05) (Fig. 8.23a, b). Dephosphorylation and phos-
phorylation induced by PKA of μ-calpain accelerated its degradation and activation.
Studies have shown that dephosphorylation improved the activity of μ-calpain
δAε δBε
δCε δDε
δEε δFε
Fig. 8.21 Phosphorylation of heat stable proteins during incubation with μ-calpain. (a), (b) Pro-
teins incubated with 25 units μ-calpain. (c), (d) Proteins incubated with 50 units μ-calpain. (e), (f)
Proteins incubated with 100 units μ-calpain. (g), (h) Proteins incubated with 150 units μ-calpain.
(a), (c), (e), (g) SDS-PAGE gels stained with Pro-Q Diamond. (b), (d), (f), (h) SDS-PAGE gels
stained with SYPRO Ruby. (i), (j), (k), (l) Relative phosphorylation level of proteins incubated with
25, 50, 100, 150 units μ-calpain, respectively. AP Alkaline phosphatase, PKA Protein kinase A
(Du et al. 2019)
Aε
Bε
Cε
Dε
1.5 2.0 a
δEε
ε δFε
ε
a b
1.5
1.0 c
ε AP ε 1.0 a aaa bb AP
bb
0.5 Control Control
0.5
PKA PKA
0.0 0.0
1 2 12 24 1 2 12 24
Incubation time/h Incubation time/h
2.5 a 3.0
a
δGε
ε 2.0 δHε
ε
a 2.5 a
bb bb
2.0 a
1.5 bb bb
a a AP a b AP
1.5 b
1.0 b bb
bb b Control 1.0 Control
0.5 0.5
PKA PKA
0.0 0.0
1 2 12 24 1 2 12 24
Incubation time/h Incubation time/h
Fig. 8.22 Degradation of 80 kDa μ-calpain subunit during incubation. (a–d) The degradation of
80 kDa μ-calpain subunit incubated with 25 (a), 50 (b), 100 (c), 150 units (d) μ-calpain. (e–h)
Relative degradation rate of 80 kDa μ-calpain subunit incubated with 25 (e), 50 (f), 100 (g),
150 units (h) μ-calpain. Values with different letters differ at the same incubation time (P<0.05).
AP Alkaline phosphatase, PI Phosphatase inhibitor (Du et al. 2019)
(Du et al. 2017a). But different protein kinases functioned differently in regulating
μ-calpain activity (Storr et al. 2011; Leloup et al. 2010). In the calpastatin-free
control system in present study, PKA phosphorylated μ-calpain also enhanced the
activity of μ-calpain, which was consistent with the results of our recent research
(Du et al. 2017b, 2018). However, μ-calpain in PKA treatment showed significantly
lower degradation rate than control at 24 h in 50 units μ-calpain incubation system
(P < 0.05) (Fig. 8.22f). Besides, in 100 and 150 units μ-calpain incubation system,
there was no significant difference in the degradation rate of μ-calpain between PKA
Aε
Bε
2.5
2.0 a a
a b
a
1.5
b AP
1.0 Control
0.5 PKA
0.0
1 2
Incubation time/h
Cε
Fig. 8.23 Western blotting analysis and casein zymography analysis of μ-calpain incubated in
absence of calpastatin. (a) Detection of degradation of 80 kDa μ-calpain subunit by western blot. (b)
Relative degradation rate of 80 kDa μ-calpain subunit. (c) Casein zymography analysis of μ-calpain.
Values with different letters show significant difference in the results in different groups at the same
incubation time (P < 0.05). AP Alkaline phosphatase, PI Phosphatase inhibitor (Du et al. 2019)
and control treatments during incubation (P > 0.05) (Fig. 8.22g, h). These results
suggested that the degradation of PKA phosphorylated μ-calpain could probably be
easily inhibited by calpastatin.
Casein zymography detects the native and autolyzed μ-calpain which has the
proteolytic activity to degrade casein in gels. Casein zymography analysis of
μ-calpain incubated without calpastatin is shown in Fig. 8.23c. The top row of
bands was native μ-calpain, the bottom row of bands was autolyzed μ-calpain. The
results showed that μ-calpain in AP and PKA groups autolyzed faster than that of
control. After 24 h incubation, μ-calpain was still detected in control group but not in
AP and PKA groups, indicating that without calpastatin, dephosphorylation and
PKA phosphorylation positively regulated μ-calpain degradation/autolyzation.
When 25 units of μ-calpain were added to heat stable proteins, the autolysis of
Fig. 8.24 Casein zymography analysis of μ-calpain incubated with calpastatin. (a), (b), (c), (d)
25, 50, 100, 150 unit μ-calpain was incubated with heat stable proteins, respectively. AP Alkaline
phosphatase, PKA Protein kinase A (Du et al. 2019)
μ-calpain was prevented and the intensity of intact μ-calpain was not different among
the three treatments (Fig. 8.24a). As the content of μ-calpain added to heat stable
proteins increased, the autolyzed μ-calpain was detected in casein gels (Fig. 8.24b, c,
d). In 50 and 100 units μ-calpain incubation systems, the intensity of intact μ-calpain
in AP group decreased faster than in the other two groups. Besides, the amount of
autolyzed μ-calpain was higher in control group than that of PKA group at 24 h
incubation. But when the incubation system contained 150 units of μ-calpain, the
autolysis status of μ-calpain in control and PKA groups showed no difference. These
results were consistent with the degradation rate of μ-calpain measured by western
blotting. Increased degradation and activity of PKA phosphorylated μ-calpain were
limited by calpastatin.
Previous studies have reported that dephosphorylation of μ-calpain enhanced its
activity (Du et al. 2017a, b). However, phosphorylation of μ-calpain induced by
different kinases may have opposite effects. Protein kinase Cɩ (PKCɩ) induced
μ-calpain and m-calpain phosphorylation enhanced their activity and was related
to increased cell migration (Xu and Deng 2006a). In the meantime, m-calpain
phosphorylated by PKA reduced its activity by blocking the binding sites of
m-calpain to its activator (Leloup et al. 2010; Shao et al. 2006). Calpain phosphor-
ylated by different protein kinases usually occurred at different amino acid residues,
accounting for the different roles they played in regulating calpain activity (Storr
et al. 2011). Our recent study showed that increased phosphorylation level of
sarcoplasmic proteins induced by phosphatase inhibitor (PI) inhibited μ-calpain
activity (Du et al. 2017a). PKA was also reported to positively regulate μ-calpain
activity (Du et al. 2017b). Both AP and phosphatase inhibitor had broad-spectrum
ability to decrease or increase the phosphorylation level of proteins. Phosphatase
inhibitor improved the phosphorylation of proteins by inhibiting dephosphorylation
process rather than working directly on protein kinases, indicating that the overall
effects of phosphorylation induced by all protein kinases on μ-calpain activity might
be negative. Besides, in the present study, calpastatin had higher inhibitory ability on
the activity of PKA phosphorylated μ-calpain, which might be another reason why
PI treated sarcoplasmic proteins showed lower μ-calpain activity.
8.7.4 The Degradation of Calpastatin
The undegraded calpastatin in lamb longissimus muscles is about 130 kDa in size
and degrades gradually to small fragments during postmortem storage (Doumit and
Koohmaraie 1999). The discrete degradation products of calpastatin include immu-
noreactive peptides about 100, 80, and 50 kDa. In 25 units μ-calpain system, there
were no distinct changes in calpastatin in AP, control, and PKA groups during
incubation. As the amount of μ-calpain in incubation system increased, calpastatin
degradation increased. The 130, 100, and 80 kDa calpastatins were no longer
detectable in all three treatments when 150 units of μ-calpain were added
(Fig. 8.25d). Apparently, calpastatin in AP group degraded much faster than control
and PKA groups in 100 and 150 units μ-calpain incubation systems (Fig. 8.25c, d).
In addition, PKA group presented the lowest degradation rate of calpastatin when
samples were incubated with 50 units of μ-calpain (Fig. 8.25b). Proteolytic enzymes,
such as μ-calpain, m-calpain, cathepsin, and proteasome, degraded calpastatin, but
μ-calpain was the major contributor to this process in postmortem muscles (Doumit
and Koohmaraie 1999; Huang et al. 2014). Thus, the μ-calpain added to the systems
was not only inhibited by calpastatin but also degraded calpastatin in turn. Therefore,
the relatively stable state of calpastatin during incubation with 25 units of μ-calpain
could be illustrated by the greatly suppressed and indifferent μ-calpain activity in the
three treatments. Meanwhile, the results of calpastatin degradation in 50, 100, and
150 units μ-calpain incubation systems agreed well with the corresponding activity
of μ-calpain. Calpastatin in skeletal muscle contained four repeated calpain-
inhibiting domains (Lee et al. 1992). The ability of μ-calpain to degrade calpastatin
reduced activity of calpastatin, but cannot completely remove the inhibition of
μ-calpain by calpastatin (Demartino et al. 1988; Doumit and Koohmaraie 1999;
Koohmaraie et al. 1990). According to the results of μ-calpain activity in calpastatin-
free system, PKA treated μ-calpain degraded more calpastatin than control. Consid-
ering μ-calpain activity in PKA treatment was extensively inhibited by calpastatin,
Fig. 8.25 Degradation of calpastatin during incubation. (a), (b), (c), (d) 25, 50, 100, 150 unit
μ-calpain was incubated with heat stable proteins, respectively. AP Alkaline phosphatase, PKA
Protein kinase A (Du et al. 2019)
the weakened proteolytic ability of μ-calpain contributed to the less degradation of

calpastatin than in the other two treatments in 50 units μ-calpain incubation system.
Consequently, PKA phosphorylated μ-calpain had stronger inhibitory effects on
calpastatin.
8.8 Conclusions
Broad-spectrum catalyzing phosphorylation and dephosphorylation played negative

or positive roles in regulating μ-calpain activity, respectively. However, phosphor-
ylation induced by PKA enhanced the activity of μ-calpain. Meanwhile, PKA
References 187
phosphorylated μ-calpain was more sensitive to calpastatin and had stronger inhib-
itory effects on calpastatin. Therefore, although PKA phosphorylation enhanced
μ-calpain activity, phosphorylation negatively regulated the activity of μ-calpain in
general. The mechanism of the effects of phosphorylation on μ-calpain activity was
investigated in this study, which helped us to better understand the influencing
mechanism of μ-calpain activity and meat tenderness.
Acknowledgments Parts of this chapter are reprinted from Food Chemistry, 228, Du, M., et al.
Phosphorylation inhibits the activity of μ-calpain at different incubation temperatures and Ca2+
concentrations in vitro, 649–655; Food Research International, 100, Du, M., et al. Effects of
phosphorylation on μ-calpain activity at different incubation temperature, 318–324; Food Chemis-
try, 252, Du, M. et al., Phosphorylation regulated by protein kinase A and alkaline phosphatase play
positive roles in μ-calpain activity, 33–39. Food Chemistry, 274, Du, M., et al. Calpastatin inhibits
the activity of phosphorylated μ-calpain in vitro, 473–479. Copyright (2020), with permission from
Elsevier.
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Chapter 9
Phosphorylation on Myoglobin
Abstract As the most important pigment for meat color, myoglobin mainly exists
in sarcoplasm, and meat color is determined by its absolute content as well as the
relative content of three types of myoglobin (oxymyoglobin, deoxymyoglobin, and
metmyoglobin). Previous studies have shown that protein phosphorylation may
negatively regulate the stability of meat color through the regulation of glycolysis
pathway and myoglobin. The purpose of this research was to study the influence of
phosphorylation on myoglobin stability, in order to provide a theoretical basis for
improving the stability of meat color through regulating phosphorylation level.
Sodium hydrosulfite was used to reduce myoglobin from skeletal muscle, and later
was removed by ultrafiltration. Then alkaline phosphatase (AP) was added to in vitro
incubation system to catalyze the dephosphorylation of myoglobin. The changes in
myoglobin phosphorylation level were measured by SDS-polyacrylamide gel elec-
trophoresis (SDS-PAGE) and Pro-Q Diamond and Ruby phosphoprotein gel
staining. We also kept a record of the changes in pH and used ultraviolet spectro-
photometer to measure the relative content of three myoglobin redox forms. The
ultraviolet spectrophotometer of myoglobin was determined by circular dichroism
(CD) spectroscopy. Myoglobin phosphorylation may lead to changes in its second-
ary structure, therefore reducing myoglobin stability and increasing its autoxidation
rate, which further accelerated the accumulation of metmyoglobin. As a result, meat
color was found to have a low stability, and this might be one of the explanations that
protein phosphorylation negatively regulated meat color stability.
Keywords Meat color · Myoglobin · Protein phosphorylation · Redox stability ·

Secondary structure
9.1 Introduction
The most intuitive impression of consumers towards meat and meat products comes
from meat color. Meat with poor color gives people the feeling of unsanitary and
poor quality; therefore, meat color is one of the most important factors affecting
consumers’ purchase decisions. Myoglobin is the most important pigment in

https://doi.org/10.1007/978-981-15-9441-0_9
192 9 Mechanism of the Effect of Protein Phosphorylation on Myoglobin
postmortem meat. Studying the influencing factors of myoglobin stability can

provide a theoretical basis to improve meat color stability.
Canto et al. (2015) found out that an acid transfer happened to myoglobin
isoelectric point, which indicated that there was a post-translation modification in
myoglobin (PI ¼ 6.78), and the content of the modified point of myoglobin was high
in the unstable meat color group. Thus, we could say that this kind of post-translation
modification in myoglobin would affect meat color stability. It has been proved that
the phosphorylation modification existed in human and mouse myoglobin (www.
photosite. ORG). Therefore, the speculation was that phosphorylation modification
in myoglobin might affect meat color stability. Previous research from our team
showed that protein phosphorylation could negatively regulate meat color stability,
and the possible pathway might be through regulating the glycolysis process and
myoglobin stability.
At present, there are few studies about the effect of protein phosphorylation on
meat color stability, and neither much on the effect of phosphorylation on myoglobin
stability. Based on the pure myoglobin from skeletal muscles, we used sodium
dithionite as reductant to obtain deoxymyoglobin. And the incubation of myoglobin
and alkaline phosphatase (AP) was conducted in vitro at 37 C to regulate myoglobin
phosphorylation level and obtain myoglobin samples with different phosphorylation
level. We studied the relationship between myoglobin phosphorylation and its redox
stability, and clarified the effect of phosphorylation on the secondary structure of
myoglobin.
9.2 Changes of Myoglobin Relative Contents in Meat After

Regulation of Protein Phosphorylation
Myoglobin is the most important pigment affecting meat color. It mainly exists in the
sarcoplasm. The absolute content of myoglobin and the relative content of the three
kinds of myoglobin (oxymyoglobin, deoxymyoglobin, and metmyoglobin) deter-
mine the meat color. The influence of protein phosphorylation on meat color stability
was investigated in Chap. 2. Phosphatase and protein kinase inhibitors were added to
minced ovine Longissimus thoracis et lumborum (LTL) muscle to adjust the global
phosphorylation of sarcoplasmic proteins. (1) Phosphatase inhibitor group (high
phosphorylation level), muscle pieces were added with phosphatase inhibitor
PhosStop (Roche, Mannheim, Germany, one tablet per 5 g muscle pieces), which
was dissolved in saline to inhibit the dephosphorylation of proteins; (2) Saline
control group, muscle was added with the vehicle of saline; (3) Kinase inhibitor
group (low phosphorylation level), muscle was added with kinase inhibitors
(M2066, Abmole, Houston, TX, USA, 0.144 lmole per 5 g muscle pieces), which
were dissolved in dimethyl sulfoxide (DMSO) and saline to inhibit the activity of
protein kinases; (4) DMSO control group, muscle was added with DMSO and saline.
After adding the reagents, muscles were minced and stored at 4 C for 7 days. All the
9.2 Changes of Myoglobin Relative Contents in Meat After Regulation of Protein. . . 193
operations were done within 40 min after exsanguination. Minced muscle samples
were collected from all four groups at 2 h, 24 h, 2 days, 3 days, 5 days, and 7 days
postmortem, snap frozen in liquid nitrogen, and stored at 80 C until analysis. The
data obtained showed that the rate and extent of pH decline, along with lactate
accumulation in postmortem muscle, were related to protein phosphorylation. Anal-
ysis of meat color stability and the relative content of myoglobin redox forms
revealed that meat color stability was inversely related to the phosphorylation of
sarcoplasmic proteins. Thus, this study suggested that protein phosphorylation may
be involved in meat color development by regulating glycolysis and the redox
stability of myoglobin.
Meat color is affected by the proportions of three myoglobin forms, which are
influenced by the oxygen availability, the autoxidation rate of myoglobin (Fe2+), and
the reducing capacity of metmyoglobin (MetMb) (Mancini and Hunt 2005). Oxida-
tion of deoxymyoglobin (DeoxyMb) and oxymyoglobin (OxyMb) to MetMb leads
to brown discoloration. The relative content of DeoxyMb and MetMb in this study
was shown in Table 9.1. The relative content of OxyMb was higher (P < 0.05) in the
kinase inhibition group than in the EMSO control samples at all times, except for
120 h. The relative content of OxyMb showed no difference between Saline control
and DMSO control groups. Compared with the other three groups, the relative
content of OxyMb was the lowest (P < 0.05) in the phosphatase inhibition group
during the whole period except for 2 h, indicating a higher relative content of
OxyMb in meat with lower protein phosphorylation. The observed changes in the
relative content of OxyMb for treatment groups matched with changes of a* values.
No difference was found in the relative content of DeoxyMb between the phospha-
tase inhibition group and the Saline control group for the first 24 h, which then
differed afterwards. DeoxyMb in the meat with phosphatase inhibitor was signifi-
cantly lower than in the kinase inhibitor added meat at all times except for 2 h
(P < 0.05), indicating a higher relative content of DeoxyMb in meat of lower protein
phosphorylation. The highest (P < 0.05) relative content of MetMb was determined
in the phosphatase inhibition group all the time in the four treatments. Although no
difference in MetMb content existed between the kinase inhibition group and the
DMSO control group at 48, 120, and 168 h, higher MetMb was detected at 2, 24, and
72 h in DMSO control group (P < 0.05).
Among the four treatments, the relative content of DeoxyMb and OxyMb
decreased the fastest (P < 0.05) in the phosphatase inhibited meat, while MetMb
content increased the fastest (P < 0.05) in this group during the first 48 h and then
kept constant afterwards. MetMb in the kinase inhibited meat increased much slower
(P < 0.05) when compared with the other three groups during the first 48 h. On the
contrary, the relative content of the OxyMb in the kinase inhibition group increased
more rapidly (P < 0.05) than in the other three groups during the first 48 h and then
declined till the end of the experimental period. The changes of relative content of
different myoglobin forms were similar between Saline control and DMSO control
groups, which were intermediate among the four treatments all the time. In sum-
mary, the relative content of different myoglobin forms differed significantly
(P < 0.05) between the kinase inhibition group and phosphatase inhibition group,
194
Table 9.1 Effects of inhibitors and storage time (hour) on relative pigment content (Li et al. 2017a)
Attribute Group 2 24 48 72 120 168
Deoxymyoglobin (1.5-K/S Phosphatase 0.68 0.04aX 0.54 0.02bX 0.47 0.08cX 0.46 0.02cX 0.44 0.02c X 0.43 0.02cX
ratio) inhibition
Saline control 0.68 0.04aX 0.56 0.01bXY 0.69 0.02aY 0.57 0.01bY 0.56 0.02bY 0.56 0.02bY
DMSO control 0.68 0.05a X 0.57 0.01bY 0.68 0.03aY 0.57 0.02bY 0.57 0.02bY 0.57 0.01bY
Kinase inhibition 0.68 0.04aX 0.58 0.01bY 0.75 0.02cZ 0.57 0.01bY 0.57 0.01bY 0.57 0.01bY
Oxymyoglobin (1-K/S Phosphatase 0.59 0.04aX 0.49 0.04bX 0.43 0.05cX 0.44 0.03cX 0.40 0.04cX 0.39 0.01cX
ratio) inhibition
Saline control 0.61 0.02aX 0.61 0.02aYZ 0.61 0.03aY 0.57 0.02bY 0.56 0.02bY 0.55 0.02bYZ
DMSO control 0.59 0.02aX 0.59 0.03abY 0.60 0.03aY 0.56 0.02bcY 0.55 0.02cY 0.53 0.03cY
Kinase inhibition 0.61 0.02aX 0.64 0.03bZ 0.68 0.02cZ 0.61 0.01aZ 0.58 0.01dY 0.56 0.02dZ
Metmyoglobin (2-K/S Phosphatase 0.80 0.02aX 1.20 0.06bX 1.28 0.04cX 1.31 0.03cX 1.29 0.03cX 1.28 0.01cX
ratio) inhibition
Saline control 0.77 0.02aYZ 1.00 0.04bY 1.16 0.02cY 1.07 0.05dY 1.09 0.05dY 1.11 0.05dY
DMSO control 0.78 0.01aY 0.97 0.05bY 1.16 0.04cY 1.08 0.05dY 1.09 0.05dY 1.10 0.04dY
Kinase inhibition 0.75 0.01aZ 0.87 0.04bZ 1.11 0.03cY 1.01 0.04dZ 1.07 0.03eY 1.12 0.04cY
DeoxyMyoglobin, OxyMyoglobin, and MetMyoglobin content are evaluated by the K/S ratios and expressed as (1.5-K/S ratio), (1-K/S ratio), and (2-K/S ratio),
respectively. Data are presented as mean standard deviation (n ¼ 5). Data with different lowercase letters in a row are significantly different (P < 0.05). Data
with different capital letters in the same column are significantly different (P < 0.05)
9 Mechanism of the Effect of Protein Phosphorylation on Myoglobin
9.2 Changes of Myoglobin Relative Contents in Meat After Regulation of Protein. . . 195

3.5 DMSO control kinase inhibition
ab ab b
3.0 a
c
2.5
b
R630/580
b
2.0
a c
c
1.5 bb bb bbc bbb
a a a a
1.0
0.5
0.0
2 24 48 72 120 168
Fig. 9.1 Effects of protein phosphorylation on R630/580 values. Data with different letters at the
same time point are significantly different (P < 0.05). Data are presented as mean standard
deviation, n ¼ 5 (Li et al. 2017a)
indicating that the redox stability of myoglobin was influenced by the protein
phosphorylation.
The R630/580 values (Fig. 9.1) were not different between Saline control and
DMSO control groups at any time. However, the R630/580 values of the phospha-
tase inhibition group were lower than those of all other groups (P < 0.05) except for
2 h. Kinase inhibition group demonstrated greater (P < 0.05) R630/580 values than
those of the rest groups except for 2 h and 168 h. R630/580 was an indirect indicator
of meat color stability, a larger ratio indicated more redness due to either OxyMb or
DeoxyMb and greater color stability (AMSA 2012). The observed changes in the
R630/580 values for treatment groups matched well with the relative content of
DeoxyMb, OxyMb, and MetMb. This suggested that kinase inhibition group (low
phosphorylation level) was the most color-stable group, and phosphatase inhibition
group (high phosphorylation level) was most color-labile group.
All data suggested that meat color stability was significantly affected by the
protein phosphorylation/dephosphorylation. According to Canto (2015), nine pro-
teins were differentially abundant in color-stable and color-labile steaks, which
included pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, Myosin
regulatory light chain 2, and myoglobin. Joseph et al. (2012) reported that heat shock
protein-27 kDa, pyruvate dehydrogenase, and pyruvate kinase isozymes M1/M2
isoform were correlated with meat color. Those color-related proteins were identified
to be phosphorylated (Chen et al. 2016; Huang et al. 2011, 2012). In the present
study, difference of the phosphorylation level of those color-related proteins
between groups may cause the difference in meat color stability.
9.3 Changes of Myoglobin Redox Stability After Regulation

of Protein Phosphorylation In Vitro
Studies have shown that protein phosphorylation might have a negative effect on
meat color development by regulating glycolysis and the redox stability of myoglo-
bin. The effects of phosphorylation on myoglobin stability were investigated in this
study, which would provide theoretical basis for improving meat color stability
through regulating protein phosphorylation level. The pure MetMb from skeletal
muscle was used in this experiment. MetMb was reduced by sodium dithionite,
which was then removed by ultrafiltration. After that, AP was added to catalyze the
dephosphorylation of myoglobin in vitro. According to the results, the phosphory-
lation level of myoglobin was significantly lower (P < 0.05) in AP group than that in
control group at 6 h, which indicates that AP can catalyze the dephosphorylation of
myoglobin in vitro, thus decreasing the phosphorylation level of myoglobin. The
relative content of oxymyoglobin in AP group was significantly higher than that in
control group, and the relative content of MetMb was significantly lower than that in
control group after 2 h. In brief, the automatic oxidation rate of myoglobin was lower
and the redox stability of myoglobin was higher in AP group than that in control
group. However, no significant difference (P > 0.05) was observed between AP
group and control group, which meant that pH value of the incubation system was
not changed by adding AP. The results showed that the secondary structure of
myoglobin was mostly α-helix. From 0 min to 6 h incubation, the content of
α-helix and β-sheet of myoglobin was almost unchanged in AP group, while the
α-helix content of myoglobin increased and β-sheet content of myoglobin decreased
in control group, indicating that the secondary structural stability of myoglobin was
increased after dephosphorylation. It was speculated that the secondary structure of
myoglobin might be changed after phosphorylation. The secondary structure stabil-
ity was decreased and automatic oxidation rate of myoglobin was increased after
phosphorylation, thus leading to the accumulation of MetMb and color deterioration.
This might be one of the reasons by which protein phosphorylation played a negative
role in regulating meat color stability.
9.3.1 Changes of Myoglobin Phosphorylation Level
Proteins are phosphorylated by kinase catalysis and dephosphorylated by phospha-

tase catalysis. AP can catalyze the dephosphorylation of proteins and reduce the
phosphorylation level of proteins (Wang et al. 2014). The results of myoglobin
phosphorylation levels were shown in Fig. 9.2. At 6 h of incubation, there was a
significant difference in the phosphorylation level of myoglobin between the two
groups. The phosphorylation level of myoglobin in AP treatment (AP group) was
significantly lower than that of the control group (P < 0.05), indicating that AP
9.3 Changes of Myoglobin Redox Stability After Regulation of Protein. . . 197
Fig. 9.2 Gel images of phosphorylated and total myoglobin by 1-DE. (a): Image of phosphorylated
myoglobin stain with Pro-Diamond; (b): Image of total myoglobin stain with SYPRO Ruby.
(Li et al. 2017b)
0.45 AP Control
Phosphorylation level (P/T ratio)
0.40 a
b
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0min 30min 2h 6h 24h 2d
Incubation time
Fig. 9.3 Analysis of the phosphorylation level of myoglobin between AP group and control group.
Data with different letters at the same time points are significantly different between two groups
(P < 0.05). (Li et al. 2017b)
catalyzed the dephosphorylation of myoglobin and reduced the phosphorylation

level of myoglobin (Fig. 9.3).
9.3.2 Changes of Myoglobin Relative Contents
Myoglobin consists of a molecule of globin (153 amino acids) and a heme cofactor
(Kendrew 1963). The heme cofactor is composed of an iron porphyrin consisting of
an iron ion and a tetramolecular pyrrole ring. There are six coordination bonds for
iron ions, of which the sixth one can bind to some small molecules, such as oxygen,
nitrogen, water, carbon monoxide, etc. Due to the different ligands bound by the
sixth ligand of myoglobin, myoglobin exists in many states, including DMb,
OxyMb, and MetMb. Myoglobin is the most important pigmentation substance in
meat with sufficient bleeding after slaughter, and the absolute content of myoglobin
and the relative content of three kinds of myoglobin determine the meat color
(Bekhit and Faustman 2005). The relative contents of the three kinds of myoglobin
were shown in Fig. 9.4.
At 0–30 min, DeoxyMb was the main component of the incubation system, and
then DeoxyMb combined with oxygen to produce oxymyoglobin. At the same time,
myoglobin was oxidized to produce MetMb. When incubated for 2 h, OxyMb and
MetMb were dominant in the system. The content of OxyMb in the system
decreased gradually and the content of MetMb increased gradually from 2 h. The
relative content of DeoxyMb, OxyMb, and MetMb in the incubation system was
significantly different between AP group and control group from 2 h after incubation
(P < 0.05). Among them, the content of OxyMb in control group was
significantly lower than that in AP group (P < 0.05), the content of DeoxyMb was
significantly lower than that in AP group (P < 0.05), and the content of MetMb was
significantly higher than that in AP group (P < 0.05). This indicated that compared
with the control group, the content of reduced myoglobin in AP group was higher
(P < 0.05), and the content of MetMb was lower (P < 0.05).
9.3.3 Changes of pH in Myoglobin Incubation System
pH is an important factor affecting the stability of myoglobin. In order to determine

whether the pH of incubation system has an effect on the stability of myoglobin, the
changes of the pH of incubation system during incubation were measured. The
results showed that there was no significant difference in the pH between AP
group and control group during the whole incubation process (P > 0.05)
(Fig. 9.5). This indicated that the addition of AP did not change the pH of incubation
system. Therefore, it was speculated that the difference of myoglobin stability
between AP group and control group during incubation was not caused by pH.
Fig. 9.4 Changes of the

relative proportions of
a
deoxymyoglobin, 1.2 Control
Relative content of OxyMb

oxymyoglobin, and
1.0 AP
metmyoglobin over time. a
(a): Changes of the relative a
proportions of 0.8
oxymyoglobin over time; b
(b): Changes of the relative 0.6
proportions of
deoxymyoglobin over time; 0.4 b
a
(c): Changes of the relative
0.2 b b a
proportions of
metmyoglobin over time.
Data with different letters at 0.0
the same time points are 0min 30min 2h 6h 24h 48h
significantly different Incubation time
between two groups
(P < 0.05) (Li et al. 2017b)
b
Relative content of DeOxyMb
1.2
Control
1.0 AP
0.8
0.6
0.4
0.2 ba ba
0.0
0min 30min 2h 6h 24h 48h
(0.2)
Incubation time
c
1.0 a a
Relative content of MetMb
b
Control b
0.8 a
AP
0.6
a b
0.4
b
0.2
0.0
0min 30min 2h 6h 24h 48h
Incubation time
7.50
7.45
7.40 Control AP
7.35
7.30
pH
7.25
7.20
7.15
7.10
7.05
7.00
0h 30min 2h 6h 24h 48h
Incubation time
Fig. 9.5 Changes of the pH values in the incubation system over time. Data with different letters at
the same time points are significantly different between two groups (P < 0.05) (Li et al. 2017b)
Table 9.2 Changes of myoglobin secondary structure (Li et al. 2017b)

Time
0 min 30 min 6h 24 h 48 h
α-helix (%) AP 95.2 95 95.2 100 100
Control 95.2 96.3 98.5 100 100
β-fold (%) AP 4.9 5.03 4.56 0 0
Control 4.9 3.75 1.3 0 0
9.3.4 Changes of Myoglobin Secondary Structure
The secondary structure of myoglobin is mainly α-helix (Bisig et al. 1995). Changes
of secondary structure of myoglobin before and after incubation with AP were
shown in Table 9.2. The secondary structure of myoglobin was mainly α-helix.
When incubated for 30 min and 6 h, the content of α-helix in AP group was lower
than that in control group, and the content of β-fold was higher than that in control
group. At 24 and 48 h of incubation, the secondary structure of myoglobin in AP
group and control group was mainly α-helix, and there was no difference in helix
content between the two groups. From 0 to 6 h after incubation, the content of
α-helix and β-fold of myoglobin in AP group changed slightly. The content of
α-helix of myoglobin in control group increased and the content of β-fold decreased.
This indicated that the secondary structure stability of myoglobin in AP group was
higher than that in control group. It means that the secondary structure of myoglobin
was affected by phosphorylation of myoglobin, and the secondary structure stability
of myoglobin at low phosphorylation level was higher.
When DeoxyMb was dominant on the surface of meat, the meat was purple red or
purple pink. When the surface of meat was mainly oxymyoglobin, the meat appeared
to be bright red. When the surface of meat was predominantly MetMb, the meat was
brown, so the production of MetMb was accompanied by the deterioration of meat
color (Suman et al. 2007). The content of MetMb in AP group was significantly
lower than that in control group, indicating that the automatic oxidation rate of
myoglobin in AP group was lower than that in control group, and the redox stability
was higher than that in control group. In addition, the phosphorylation level of
myoglobin in AP group was lower than that in control group. It was speculated that
the auto-oxidation rate of myoglobin in low phosphorylation level group was lower
than that in high phosphorylation level group. It means that the redox stability of
myoglobin at low phosphorylation level was higher than that of myoglobin at high
phosphorylation level, which might negatively regulate the redox stability of
myoglobin.
The declined rate and degree of pH postmortem is one of the most important
factors affecting meat color (Scheffler and Gerrard 2007), and the redox rate of
myoglobin is significantly affected by pH. The stability of globin and hemoglobin
covalent binding bond in myoglobin molecule could be reduced at lower pH, which
would increase the automatic oxidation rate of myoglobin and reduce the oxidation-
reduction stability and make it easy to be oxidized. Gutzke and Trout (2002) found
out that the oxidation rate of myoglobin was significantly affected by pH at different
species and temperature. Shikama and Sugawara (1978) studied the effect of pH
(4.8–2.6) on myoglobin oxidation. The results showed that the oxidation of myo-
globin was directly affected by H+ concentration. In this study, the pH of incubation
system between AP group and control group had no difference, so the effect of pH
on myoglobin stability could be excluded. That is, the difference of myoglobin
stability between AP group and control group was not caused by pH. In addition,
since incubation tests were conducted with the same materials and at the same
ambient temperature, the effect of other external factors on the rate of myoglobin
autoxidation could be excluded. It was speculated that the difference in stability
between AP group and control group was due to the difference in phosphorylation
level of myoglobin between the two groups.
Myoglobin is a monomer globulin that stores oxygen and transmits oxygen to
mitochondria. Studies have shown that covalent or non-covalent modification was a
common phenomenon to regulate the activity of monomer globulin (Ascenzi et al.
2013). For example, human neuroglobulin (monomer globulin) can be phosphory-
lated (covalent modification) to protect nerve cells under hypoxic conditions both
in vivo and in vitro. It is speculated that the phosphorylation of myoglobin may be
related to oxygen content, and alters the binding ability of myoglobin to oxygen,
which in turn affects the existence of myoglobin. Studies have shown that phos-
phorylation can regulate the structure, activity, and stability of proteins. For exam-
ple, PK can be transformed into an acid-stable isomer after phosphorylation, and
then maintain high activity in PSE meat (Schwägele et al. 1996). The serine at the
14th position of GP can be phosphorylated, causing structural changes and activa-
tion (Sprang et al. 1988; Johnson 1992). After phosphorylation, fructokinase phos-
phate forms a complex with actin, which regulates the activity of fructokinase
phosphate and provides energy for cells (Cai et al. 1997; Kuo et al. 1986). In
addition, study by Diaz-Moreno et al. (2009) has shown that phosphorylation of
KH-type splicing regulatory protein (KSRP) can extend the irregular structure in the
unstable KH1 region, thereby altering the structure of the protein. Zhang and Liu
(2017) showed that serine of (Gossypium hirsutum pyruvate kinase 6) GhPK6 was
phosphorylated at positions 215 and 402. Phosphorylation of serine at positions
215 inhibited the activity of the enzyme, and phosphorylation at positions 215 and
402 promoted the degradation of the protein. In this study, the secondary structure
stability of myoglobin with different phosphorylation levels was also different.
Therefore, it is speculated that the difference of phosphorylation level of myoglobin
between AP group and control group may lead to the difference of secondary
structure of myoglobin, and the difference of structure may lead to the difference
of automatic oxidation rate and redox stability of myoglobin. After phosphorylation
or dephosphorylation of myoglobin, it may regulate its secondary structure, and then
affect the binding state of myoglobin with oxygen, and ultimately affect the auto-
matic oxidation rate and redox stability of myoglobin.
9.4 Conclusions
AP could catalyze dephosphorylation of myoglobin in vitro which decreased the

phosphorylation level of myoglobin. The autoxidation rate of myoglobin decreased
in AP treated group, i.e. the redox stability of myoglobin increased. The pH of the
incubation system was not influenced by adding AP which excluded the possible
effects of pH on myoglobin stability. The secondary structure of myoglobin
increased after dephosphorylation compared with control group. According to the
results from this study, it can be concluded that phosphorylation of myoglobin could
decrease its stability of secondary structure and result in decreased myoglobin redox
stability and meat color, and this could be a possible mechanism that protein
phosphorylation negatively regulated meat color stability.
Acknowledgments Parts of this chapter are reprinted from Food Chemistry, 219, Li, M., et al.,
Effects of protein phosphorylation on color stability of ground meat, 304-310, Copyright (2020),
with permission from Elsevier. Parts of this chapter are translated from Scientia Agricultura Sinica,
50(22), Li, M., et al., Effect of phosphorylation level on myoglobin stability (Chinese), 4382-4388.
Copyright (2020), with permission from Journal of Scientia Agricultura Sinica.
References 203
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Part III
Improvement of Meat Quality
by Regulating Protein Phosphorylation
Chapter 10
Effects of Temperature on Protein
Phosphorylation
Abstract Extensive studies have revealed that protein phosphorylation plays an

important role in postmortem meat quality, such as tenderness, color, and water
holding capacity. During storage of meat, the storage temperature is one of the most
important factors that determine meat quality. High temperature promotes meat
spoilage by accelerating biochemical reactions and microbial growth. However,
whether storage temperature affects the development of meat quality through phos-
phorylation is not well understood. In order to investigate it clearly, the postmortem
ovine muscle samples were stored at 25 C, 15 C, 4 C, and 1.5 C from 0.75 h to
21 days. The correlation between temperature, pH, adenosine triphosphate (ATP)
content, and phosphorylation levels was analyzed, and then differential phosphory-
lated proteins were identified by using liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) and gene ontology analysis. Myosin binding protein C,
troponin T3, actinin, myosin light chain, heat shock protein 90, glucose-6-phos-
phate, pyruvate kinase, enolase, fructose-bisphosphate aldolase, L-lactate dehydro-
genase, etc. were phosphorylated in postmortem ovine muscles. Moreover, ATP
content was a vital factor which had the strongest correlation with the phosphory-
lation level of individual protein band. In order to prove whether ATP content
mainly affected phosphorylation level, ATP was added to ground ovine muscle
in vitro, which showed that ATP improved the phosphorylation level of myofibrillar
proteins. The phosphorylation reaction was reversible. However, ATP was unnec-
essary for dephosphorylation. Whether the particular temperature and pH (5.2, 5.8,
6.4) had effect on the dephosphorylation of myofibrillar protein through alkaline
phosphatase are need to be studied. The result showed that 1.5 C and pH 5.2
restrained the activity of alkaline phosphatase and decreased phosphorylation level.
Phosphorylation was an essential post-translational modification and more insights
were needed for the complicated formation in postmortem muscle.
Keywords Temperature · pH · ATP content · Phosphorylation ·

Dephosphorylation · Myofibrillar proteins · Sarcoplasmic proteins · Alkaline
phosphatase · Postmortem muscle

https://doi.org/10.1007/978-981-15-9441-0_10
208 10 Effects of Temperature on Protein Phosphorylation
10.1 Introduction
Protein phosphorylation is one of the most important post-translational modifica-

tions in organisms. Recently, some researches have found out that phosphorylation
or dephosphorylation regulates the traits of meat quality, such as tenderness, color,
and water holding capacity (D’Alessandro and Zolla 2013; D’Alessandro et al.
2012a; Gao et al. 2017; Li et al. 2017; Li et al. 2018). Besides, the storage
temperature is the direct factor that contributes to the shortening, rigor, and aging
of postmortem muscle (Fernandez and Tornberg 1994; Kim et al. 2014). Thus, the
mechanism of how storage temperature regulates meat quality by protein phosphor-
ylation needs further study.
The reactions of phosphorylation and dephosphorylation are catalyzed by protein
kinase and phosphatase, respectively. With the presence of phosphate group pro-
vided by ATP, proteins are phosphorylated under the catalysis of protein kinases.
And the reversible reaction is catalyzed by phosphatase. Besides, the temperature
and pH both can affect the activity of protein kinases and phosphatases. But in
postmortem muscle, which one of temperature, pH, and/or ATP mainly affects
protein phosphorylation level is still unclear. According to the result of Ren et al.
(2020), ATP might be more related to individual protein band, although the global
phosphorylation level of sarcoplasmic proteins was stable. With ATP depletion, the
phosphorylation will be restrained, but how dephosphorylation changes with differ-
ent temperatures and pH remains unknown. In the complex system of postmortem
muscle, a more comprehensive understanding of phosphorylation or dephosphory-
lation can help us better understand the development of muscle to meat.
10.2 Effects of Temperature on Protein Phosphorylation

in Postmortem Muscle
Postmortem ovine muscle was stored at 25 C (hot fresh meat is commonly sold at
this temperature in China), 15 C, 4 C, and 1.5 C (super chilling temperature).
From the correlation analysis of temperature, pH, ATP content, and phosphorylation
level, eight myofibrillar protein bands and five sarcoplasmic protein bands were
selected for LC-MS/MS.
10.2.1 Effect of Temperature on Glycolysis in Postmortem

Muscle
The rate and extent of pH decline, along with metabolism in postmortem, were vital
for meat quality (Gratacos-Cubarsi and Lametsch 2008). The pH values of muscles
stored at different temperatures are presented in Fig. 10.1a. Generally, pH declined
10.2 Effects of Temperature on Protein Phosphorylation in Postmortem Muscle 209
（A）
7.0 25°C 15°C 4°C -1.5°C
a
aa a b
bb W
ab W W
c bc bcd bcd bc W
6.0 c c cde W
W
X de e
X X de de
pH
X de
Yd d d X X
d d
Y XY Y d d
dd
Y
d d
d
5.0
4.0
0.75 h 2 h 6h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time
（B） 3.0
25°C 15°C 4°C -1.5°C
W
Lactate (mmol/g protein)
WW W W
WW XW a
2.0 W a aX aa b Xa X b b X X
W W
c
Y X b d c c d d
b c c
XX e e X
W
cdY d
W c XY Y
WWX
X df g f
d ee h
1.0
0.0
0.75 h 2 h 6h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time
Fig. 10.1 The pH values (a) and lactate content (b) in ovine muscles stored at different temper-
atures. Data are means standard deviation (n ¼ 4). Different lowercase letters represent signif-
icant difference between different time points of the same storage temperature (P < 0.05). Different
capital letters represent significant difference between different storage temperatures at the same
time point (P < 0.05) (Ren et al. 2020)
quickly at early stage and later kept relatively constant postmortem. For muscles
stored at 4 C and 1.5 C, pH was higher than those stored at 25 C and 15 C at
12 h, 1 day, and 2 days (P < 0.05). This result suggested higher temperature
promoted glycolysis, contributing to fast pH decline (Scheffler and Gerrard 2007).
The ultimate pH of 1.5 C muscle was significantly higher than that of the other
three treatments (P < 0.05) and reached ultimate pH later than others. These data
demonstrated that 1.5 C restrained glycolysis compared with other storage
temperatures.
In postmortem muscle, anaerobic glycolysis was the major reason to decrease pH
by generating lactate and H+ (Huang et al. 2011). Lowering pH value could enhance
the effect of lactate in inhibiting glycolysis (Liu et al. 2015). The lactate content first
increased and then decreased slightly, which was consistent with the change of pH
value. Compared with other temperatures, the lactate content of 1.5 C was
significantly lower within 2 days and higher after 3 days (P < 0.05, Fig. 10.1b).
The lactate content increased and reached the maximum value in 12 h at 25 C and
15 C. However, at 4 C and 1.5 C, the highest lactate content appeared in 2 days
and 5 days, respectively, which was later than 25 C and 15 C. This result indicated
that the rate of lactate accumulation was faster in high temperature and low temper-
ature inhibited lactate accumulation by limiting glycolysis.

Muscle
High ATP consumption caused more metabolic flux, resulting in shortened rigor
mortis time (Savell et al. 2005). As shown in Fig. 10.2, ATP content was reduced
2.4
WW
Xa a W W 25°C
a X Xb Xb
ATP (nmol/µL)
1.6 Y bb Y
c
a Y
c 15°C
b Z
c WW 4°C
X
d c
d -1.5°C
0.8
Y W
d d W W
X e f
e X X
Y
e f f g
0
0.75 h 2 h 6h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time
Fig. 10.2 The ATP content in ovine muscle during storage. Data are means standard deviation
(n ¼ 4). Different lowercase letters represent significant difference between different time points of
the same storage temperature (P < 0.05). Different capital letters represent significant difference
between different storage temperatures at the same time point (P < 0.05). (Ren et al. 2020)
gradually, which was in agreement with previous studies (Shen et al. 2006; Wang
et al. 2013). ATP was exhausted in 1, 2, 5, and 7 days when muscles were stored at
25 C, 15 C, 4 C, and 1.5 C, respectively. At 0.75 h, the ATP content of muscles
stored at 25 C and 15 C was lower than that of muscles stored at 4 C and 1.5 C
(P < 0.05), which showed that ATP has been used up quickly in higher temperature
during early postmortem time. Some studies reported that ATP could be produced
only by anaerobic glycolysis in postmortem muscle, nevertheless, there were mul-
tiple processes to consume ATP, such as muscle contraction, transportation of ions,
and even protein phosphorylation (Hudson, 2012; Lametsch et al. 2002). Higher
temperature could promote anaerobic glycolysis, however, at the same time it would
accelerate pathways that increase ATP content. As a result, the ATP content
decreased with storage time.
10.2.3 Effect of Temperature on Global Phosphorylation

Levels in Postmortem Muscle
Dodecyl sulfate sodium salt and polyacrylamide gel electrophoresis (SDS-PAGE)

and fluorescence staining were used to detect phosphoproteins. Pro-Q Diamond and
SYPRO Ruby staining images of myofibrillar proteins and sarcoplasmic proteins are
shown in Fig. 10.3a, b. Seventeen individual myofibrillar protein bands and 20 indi-
vidual sarcoplasmic protein bands were selected to calculate global phosphorylation
level (Fig. 10.3c).
As shown in Fig. 10.3C-1, the protein phosphorylation level of myofibrillar
proteins decreased at 25 C and 15 C, which increased early stage and then
decreased when muscles were kept at 4 C and 1.5 C. The phosphorylation
level in the four groups showed significant difference at 6 h, 12 h, and 1 day
(P < 0.05). After 1 day storage at 4 C, the phosphorylation level increased to the
highest value and then decreased. Same changes happened when muscles were
stored at 1.5 C, and it kept the high level of protein phosphorylation for a longer
time. The main reason might relate to ATP content. At high temperature (25 C,
15 C), the consumption of ATP was faster, which would inhibit the phosphorylation
reaction. However, the consumption of ATP would not affect the dephosphorylation
reaction. As a result, the total phosphorylation level decreased gradually at 25 C and
15 C. Conversely, lower temperature (4 C, 1.5 C) restrained ATP consumption,
making the phosphorylation last longer, and the changes in myofibrillar proteins
phosphorylation level were different. Thus, the most important impact factor for
phosphorylation level of myofibrillar proteins might be the consumption of ATP
postmortem.
The phosphorylation level of sarcoplasmic proteins is shown in Fig. 10.3C-2.
Generally, the phosphorylation level increased at early storage time and then kept
stable. The phosphorylation level was significantly different (P < 0.05) at 1 and
2 days, with no difference (P > 0.05) at other storage time. It was interesting that the
Fig. 10.3 Quantification of protein phosphorylation in muscle during storage. A-1, gel image of
myofibrillar phosphoproteins stained with Pro-Q Diamond; A-2, gel image of myofibrillar proteins
stained with SYPRO Ruby; B-1, gel image of sarcoplasmic phosphoproteins stained with Pro-Q
Diamond; B-2, gel image of sarcoplasmic proteins stained with SYPRO Ruby. The global phos-
phorylation level of myofibrillar proteins (C-1) and sarcoplasmic proteins (C-2) in muscles stored at
different temperatures. Data are mean standard deviation (n ¼ 4). Different lowercase letters
represent significant difference between different time points of the same storage temperature
(P < 0.05). Different capital letters represent significant difference between different storage
temperatures at the same time point (P < 0.05) (Ren et al. 2020)
（C-1） 25℃ 15℃ 4℃ -1.5℃

W
1.5
Myofibrillar proteins phosphorylation
W a
X W W b
b
X a b a a ab
WWX c c W
a ac c c XYc c c bc c c
c a Y Y
a X
1 b b ab Y
d
Z b
level (P/T)
c Z
X
d
c
d
0.5
0
0.75 h 2 h 6h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time
（C-2） 25 ℃ 15 ℃ 4℃ -1.5 ℃
Sarcoplasmic proteins phosphorylation level
1.5
W
a a a abab
WWX
ab a abWX ab
abc ab ab
ab ababWXX aab
ab
b bcbcc ab ab
bab ab
ab ab
X ab ab ab
b abab ab
c b c
b
1
(P/T)
0.5
0
0.75 h 2h 6h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time

changes in phosphorylation level between myofibrillar proteins and sarcoplasmic

proteins were diverse. The system in postmortem muscle was complicated. As for
myofibrillar proteins and sarcoplasmic proteins of postmortem muscle, phosphory-
lation and dephosphorylation might happen at different time. Sarcoplasmic proteins
might have phosphorylation reaction earlier than myofibrillar proteins. It meant that
sarcoplasmic proteins would preferentially utilize ATP. Therefore, sarcoplasmic
proteins could keep total phosphorylation level stable rather than create drastic
changes like myofibrillar proteins.
10.2.4 Association between Phosphorylation Levels

and Temperature, pH, ATP Content
The association between global phosphorylation level or phosphorylation level of

individual protein bands and temperature, pH, ATP content was analyzed
(Table 10.1). The global phosphorylation level of myofibrillar proteins was nega-
tively correlated (P < 0.01) with temperature (r ¼ 0.548), while the global
phosphorylation level of sarcoplasmic proteins did not correlate (P > 0.05) with
temperature (r ¼ 0.277). There was no significant relation (P > 0.05) between global
phosphorylation and pH, ATP content for either myofibrillar proteins or sarcoplas-
mic proteins. As for myofibrillar proteins, the phosphorylation level of bands 13, 14
was positively correlated (P < 0.05) with temperature and ATP content, but not
(P > 0.05) with pH value. The phosphorylation level of bands 5, 7, 10, 12, 13 and
14 was positively correlated (P < 0.05) with ATP content. In terms of sarcoplasmic
proteins, the phosphorylation level of bands 3, 5, 7, 8, 10, 11, 12, 13, 14, 18, and
19 was only related to pH and ATP content (P < 0.05). The association analysis of
individual bands suggested that temperature, pH, and ATP content had different
influences on protein phosphorylation.
According to association analysis, bands 3, 4, 5, 7, 12, 13, 14, and 15 of
myofibrillar proteins and bands 10, 11, 12, 14, and 18 of sarcoplasmic proteins
were selected and subjected to protein identification.
10.2.5 Identification of Individual Protein Bands
The 8 myofibrillar protein bands and five sarcoplasmic protein bands were chosen
and identified by LC-MS/MS (Table 10.2). The proteins with unique peptide count
2 were selected for analysis. Gene ontology (GO) analysis was used to classify
these proteins into biological process (BP), molecular function (MF), and cellular
component (CC) groups.
As for myofibrillar proteins, myosin binding protein C (slow type), myosin
binding protein C (fast type), and myomesin 2 were identified in band 3. TPM1,
Table 10.1 Pearson correla- P/T ratio Temperature pH ATP content

tion coefficients of the tem-
(A)
perature, pH, and ATP content
with global phosphorylation Global 0.548** 0.162 0.004
levels Band 1 0.01 0.157 0.098
Band 2 0.414 0.338 0.323
Band 3 0.608** 0.396* 0.023
Band 4 0.166 0.409* 0.485**
Band 5 0.318 0.436* 0.468**
Band 6 0.429* 0.338 0.6**
Band 7 0.479** 0.134 0.345*
Band 8 0.068 0.361* 0.253
Band 9 0.231 0.155 0.092
Band 10 0.297 0.339 0.57**
Band 11 0.328 0.264 0.236
Band 12 0.203 0.466** 0.709**
Band 13 0.523** 0.219 0.503**
Band 14 0.417* 0.077 0.356*
Band 15 0.175 0.793** 0.658**
Band 16 0.422* 0.08 0.131
Band 17 0.735** 0.157 0.074
(B)
Global 0.277 0.113 0.211
Band 1 0.568** 0.207 0.642**
Band 2 0.252 0.29 0.515**
Band 3 0.102 0.364* 0.564**
Band 4 0.255 0.002 0.388*
Band 5 0.107 0.538** 0.434*
Band 6 0.305 0.164 0.198
Band 7 0.22 0.366* 0.532**
Band 8 0.07 0.452** 0.422*
Band 9 0.316 0.142 0.467**
Band 10 0.148 0.647** 0.699**
Band 11 0.202 0.678** 0.771**
Band 12 0.226 0.688** 0.867**
Band 13 0.088 0.355* 0.516**
Band 14 0.062 0.503** 0.682**
Band 15 0.582** 0.002 0.299
Band 16 0.347* 0.036 0.012
Band 17 0.406* 0.247 0.693**
Band 18 0.244 0.523** 0.731**
Band 19 0.402* 0.401* 0.718**
Band 20 0.066 0.309 0.49**
Note: (A) shows correlation coefficients of the temperature, pH,
and ATP content with myofibrillar proteins phosphorylation;
(B) shows correlation coefficients of the temperature, pH, and
ATP content with sarcoplasmic proteins phosphorylation; Signif-
icance levels: *, 0.01 < P < 0.05; **, P 0.01. (Ren et al. 2020)
Table 10.2 Unique proteins identified from SDS-PAGE (Ren et al. 2020)
216
Protein Peptide Unique Cover MW

number Protein name Band UniProt ID Count PepCount Percent (Da) PI
1 Myosin binding protein C, slow type 3 W5Q0I1 62 42 33.99% 126,221 6
2 Myosin binding protein C, fast type 3 W5PX04 56 38 26.82% 127,412 6.28
3 Myomesin 2 3 W5P6W4 38 35 22.66% 167,003 5.92
4 Actinin alpha 3 4 W5P9P1 101 46 42.25% 102,521 5.31
5 Actinin alpha 2 4 W5P0L8 93 42 42.52% 104,062 5.28
6 Actinin alpha 1 4 W5QJ62 27 11 10.72% 101,428 5.41
7 Actinin alpha 4 4 W5P707 24 11 11.52% 101,401 5.46
8 Heat shock protein 90 beta family member 1 5 W5Q4E1 13 13 12.66 92,678 4.77
9 Myotilin 7 W5Q1Q5 15 12 22.24% 55,667 9.11
10
10 Annexin A2 12 A2SW69 15 14 38.05% 38,612 6.92

11 Uncharacterized protein (gene 12 W5NPQ4 6 6 12.78% 36,331 6.77
name ¼ CAPZA2)
12 TPM1 13 B2LU28 10 10 28.52% 32,694 4.69
13 Four and a half LIM domains 1 protein 13 C8BKC8 11 9 22.30% 33,609 8.83
14 Four and a half LIM domains 3 13 W5QFC3 4 4 10.63% 33,341 5.93
15 Prohibitin 14 W5P7Q3 9 9 26.47% 29,804 5.57
16 14-3-3 protein gamma 14 W5PWD6 8 8 27.11% 25,736 4.76
17 Troponin T3, fast skeletal type 14 W5NRC7 7 7 19.73% 25,648 10.14
18 Troponin I type 1 variant X1 15 A0A1X9H6E3 32 16 54.55% 21,663 9.68
19 Myosin light chain 1 15 W5QD16 42 15 68.02% 21,487 5.04
20 Myosin light chain 3 15 W5PAN7 5 4 17.59% 21,931 4.99
21 Myosin light chain 6 15 W5PK27 4 3 10.75% 23,442 5.14
22 Heat shock protein family B (small) member 15 W5NUW9 3 3 13.61% 21,089 5
8
23 Glucose-6-phosphate 10 W5P323 23 14 17.95% 62,890 7.35
Effects of Temperature on Protein Phosphorylation
24 Pyruvate kinase 10 W5QC41 11 9 12.87% 61,591 8.2
10.2
25 Eukaryotic translation elongation factor 11 W5PN24 17 12 17.58% 52,215 9.28

1 alpha 2
26 Elongation factor 1-alpha 11 W5PD15 8 7 14.07% 50,140 9.1
27 Enolase 3 12 W5P663 99 25 43.09% 47,068 7.6
28 Enolase 2 12 W5P5C0 34 9 17.05% 47,240 4.94
39 Enolase 1 12 W5PIG7 17 6 12.42% 48,195 6.44
30 Fructose-bisphosphate aldolase 14 W5P1X9 87 28 60.44% 39,436 8.45
31 L-lactate dehydrogenase 14 W5PIN4 8 8 19.11% 39,742 8.58
32 Adenylate kinase isoenzyme 1 18 C5IJA8 23 12 38.14% 21,650 8.4
33 Peroxiredoxin 2 18 C8BKC5 10 6 24.24% 21,845 5.23
34 Lactoylglutathione lyase 18 W5Q0T 3 3 11.17% 21,095 5.22
Note: All protein bands were detected by LC-MS/MS spectra and analyzed by MASCOT engine against the UniProt database. Peptide Count is the number of
peptides identified by LC-MS/MS. Unique PepCount is the number of unique peptide count. Cover Percent means the number of amino acids spanned by the
assigned peptides divided by the sequence length. Molecular weight (MW) and isoelectric point (pI) are determined using Compute PI/MW tool (http://web.
expasy.org/compute_pi/). Protein number 1–22 were for about myofibrillar proteins and 23–34 were for about sarcoplasmic proteins
Effects of Temperature on Protein Phosphorylation in Postmortem Muscle
217
four and a half LIM domains 1 protein, four and a half LIM domains 3 were
identified in band 13. Band 14 contained prohibitin, 14-3-3 protein gamma, and
troponin T3 (fast skeletal type). The individual phosphorylation level of bands 3, 13
and 14 increased first and then decreased in all four groups. And those proteins in
band 3 were relevant with muscle contraction [GO: 0006936]. It meant high
phosphorylation level of those proteins in band 3 was related to muscle rigor mortis.
Meanwhile, actinin alpha 3 (band 4), actinin alpha 2 (band 4), troponin T3 (band 14),
myosin light chain 1 (band15), and myosin light chain 3 (band15) also regulated
muscle contraction (D’Alessandro et al. 2012b; Muroya et al. 2007). Bands 13 and
14, functioning in metal ion binding, were positively related to temperature and ATP
content. Actinin alpha 3, actinin alpha 2, actinin alpha 1, actinin alpha 4, heat shock
protein 90 beta family member 1, myotilin, annexin A2, uncharacterized protein
(gene name ¼ CAPZA2), troponin I type 1 variant X1, myosin light chain 1, myosin
light chain 3, myosin light chain 6, and heat shock protein family B (small) member
8 were identified in bands 4, 5, 7, 12, and 15, respectively. The changes in
phosphorylation level of these five bands were different among four different
temperatures. Heat shock protein 90 beta family member 1 is an ATP-dependent
chaperone and its phosphorylation level was positively related to ATP content.
As for sarcoplasmic proteins, glucose-6-phosphate and pyruvate kinase were
identified in band 10, and enolase 1, enolase 2, enolase 3 were identified in band
12. These proteins were glycolytic enzymes [GO: 0006096]. When muscles were
stored at 25 C and 15 C, the changes in phosphorylation level of these two bands
were inconsistent. The major reason might be that band 10 was negatively related to
pH and ATP content, while band 12 was positively related to pH and ATP content.
Researchers reported that, when glycolysis proteins were phosphorylated, some of
them regulated postmortem glycolysis and bound to muscle filaments (Sola-Penna
et al. 2010; Shen and Du 2005). Schwagele et al. (1996) showed that phosphorylated
pyruvate kinase transformed to a more stirring and acid-stable isoform. The phos-
phorylation level of pyruvate kinase was higher at 25 C and 15 C, which indicated
that phosphorylated pyruvate kinase might be related to accelerated glycolysis. The
activity of enolase 3 was associated with the regeneration and development of
muscle (Merkulova et al. 2000). Together with this finding, the phosphorylation
level of band 10 being negatively related to ATP content, it could be inferred that
high phosphorylation level of enolase 3 contributed to ATP consumption and the
transformation of muscle to meat. Eukaryotic translation elongation factor 1 alpha
2 and elongation factor 1-alpha, relating to GTPase activity [GO: 0003924], were
mainly detected in band 11. The phosphorylation level of band 11 was increased in
all muscle in the present study, which was opposite to the decreased ATP content.
Besides, fructose-bisphosphate aldolase and L-lactate dehydrogenase were identified
in band 14. Adenylate kinase isoenzyme 1, peroxiredoxin 2, and lactoylglutathione
lyase were identified in band 18. Lactoylglutathione lyase was related to apoptotic
process [GO: 0043066]. During the later stage of storage in the present study,
increased phosphorylation level of lactoylglutathione lyase might promote the
apoptotic process.
10.3 Effect of ATP on Protein Phosphorylation in Postmortem Muscle with Different. . . 219
The identified 13 individual bands were intensively related to temperature, pH,

and ATP content. And the phosphorylation level changes of individual band were
inconsistent. Myosin binding protein C, troponin T3, myosin light chain 1, heat
shock protein family B, glucose-6-phosphate, enolase 3, fructose-bisphosphate
aldolase, and other proteins were identified, and these proteins were mainly involved
in glycolysis and muscle contraction. It could be proved that storage temperature
played a vital role in biochemical process and changed phosphorylation level of
many proteins in postmortem muscle.
10.3 Effect of ATP on Protein Phosphorylation

in Postmortem Muscle with Different Temperatures
The result of Table 10.1 showed that ATP content might be the main factor affecting
phosphorylation level. In order to testify this result, the purified ATP and the same
volume of solution without ATP were added to ground meat, respectively, which
were considered as the ATP treatment group and the control group. Then ground
meat was stored at 25 C, 15 C, 4 C, and 1.5 C. The ATP content, pH, and
global phosphorylation level were determined to instigate whether ATP was a key
factor of phosphorylation.

Muscle
Energy metabolism is an important biochemical process in postmortem muscle.

ATP, a substance providing energy, has been widely reported to be correlated with
rigor mortis and glycolysis (Watabe et al. 1991; Hamm 1977). Generally, ATP
content was decreased with the increase of storage time in all treatment groups
(Fig. 10.4). After slaughtering, aerobic glycolysis stopped and anaerobic glycolysis
initiation made ATP synthesis reduce largely. The increasing consumption and
decreasing synthesis of ATP led to the total ATP content depleted in postmortem
muscle. Besides, the decomposition of ATP would affect the biochemical mecha-
nism in postmortem. Adenosine monophosphate (AMP), one of catabolites of ATP,
was linked to the activity of glycolytic enzymes. When AMP content was sufficient,
glycogen was degraded to glucose-l-phosphate by phosphorylase b. And the activity
of phosphor fructokinase (PFK) could be accelerated by AMP (Aberle and Merkel
1968).
Except for 1 day at 25 C, ATP content in ATP treated group was higher
(P < 0.05) than the control group at all time of both groups. The reason for no
significant difference of 1 d at 25 C was that ATP content could not meet the need of
ATP consumption. However, the ATP content at 1 day of 4 C was enough to
Fig. 10.4 The changes of A ATP group Control group

ATP content in ground 4.5
muscle at 25 C (a), 15 C
(b), 4 C (c), and 1.5 C
nmol/µL
Xa
Xb
3 Ya Xc
(d). Different lowercase Yb
letters represent significant Yc Xd
ATP
difference within the same 1.5
group (P < 0.05). At the Yd
e
same storage time, different 0 e
capital case letters represent 0h 0.75 h 2h 12 h 1d
significant difference Stroge time
between ATP treated group
B ATP group Control group
and the control group
(P < 0.05) 4.5
Xa
nmol/µL
Xb
Ya Xc
3 Yb Xd
Yc Xe
ATP
Yd
1.5 Xf
Ye
Yf
0
0h 0.75 h 2h 12 h 1d 2d
Storage time
C ATP group Control group

4.5
Xa Xb
Xc Xd
Ya
nmol/µL
Yb
3 Yc
Yd Xe
Xf
Ye
Xg
ATP
1.5 Xh
Yf Xi
Yf
Yg
Xj
Yh
Yi
0
0 h 0.75 h 2 h 12 h 1d 2d 3d 5d 7d 10 d
Stroge time
D ATP group Control group

4.5
Xa Xab
Xb
Ya
Yb Xc Xd
nmol/µL
3 Yc
Yd Xe Xe
Ye
Yf Yg Xf
Xg Xg
Xh
ATP
1.5
Yh
Yi Yi
Yi Xi
Yj
0
0 h 0.75 h 2 h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time
maintain ATP consumption. The ATP of 25 C and 15 C was almost completely

consumed at 1–2 days, but it still existed at 10–21 days of 4 C and 1.5 C, which
suggested ATP consumption was slower at lower temperature (Li et al. 2015)
because of repressed metabolic processes.
10.3.2 Effect of ATP on pH in Postmortem Muscle
As shown in Fig. 10.5, pH values decreased in both ATP treated group and the
control group, which was the same as Hamm (1977). The ultimate pH (pHu) value at
25 C was about 5.33 (lower than normal pHu value of intact muscle) suggested that
glycolytic rate was faster in ground muscle. Naturally, the lower pHu value appeared
at higher temperature, just as pHu(1.5 C) > pHu(4 C) > pHu(15 C) > pHu(25 C) in
this study (Kim et al. 2014). The pHu also depended on meat quality and consumer
acceptability due to pH-dependent biochemical mechanisms changed sharply in
postmortem. Aroma, flavor, juiciness, texture, and tenderness were evaluated cor-
relatively with different pHu (Devine et al. 1993).
At 25 C and 15 C, no significant difference (P > 0.05) existed between ATP
treated group and the control group during later storage time. For 4 C and 1.5 C,
the pH value of ATP treated group was higher than the control group within 12 h,
which was opposite with later time. In early storage time, there was abundant ATP in
ATP treated group which would restrain the activity of phosphofructokinase
(England et al. 2014). PFK was a key limited-speed enzyme of glycolysis through
allosteric kinetics (Costa Leite et al. 2007). The plentiful ATP would impose
negative effect on glycolysis rate and pH decline. With ATP consumption, this
kind of inhibition was gradually weakened. More H+ was produced by ATP
hydrolyzation which led to lower pH value in ATP group at later storage time.
Thus, the effect of ATP on the glycolytic enzyme activity and enhancement of ATP
hydrolyzation resulted in different pH values in ATP treated group and the control
group.
10.3.3 Effect of ATP on Global Phosphorylation Levels

in Postmortem Muscle
To estimate the effect of ATP content on myofibrillar protein phosphorylation of

different treatments, the phosphorylation level was measured through fluorescent
staining (Fig. 10.6). In the control group at 25 C and 15 C, phosphorylated proteins
divided by total proteins (P/T ratio) were reduced significantly (P < 0.05) after 12 h.
While in ATP treated group, P/T ratio reached the highest value at 2 h and later
decreased. The result demonstrated that ATP was depleted rapidly and phosphory-
lation was lower than dephosphorylation. However, the changes in P/T ratio at 4 C
A ATP group Control group

6.5
a a
X
6
b
pH Y
b c b
5.5 e c d c
5
0h 0.75 h 2h 12 h 1d
Storage time
B ATP group Control group

6.5
a a
X
6 b Y
X
b
pH
c Y X
c d Y e de e d
5.5 e
5
0h 0.75 h 2h 12 h 1d 2d
Storage time
C ATP group Control group

6.5
a a X X
6 b Y b Y
b X X X
c X X
c c X e Y c
pH
e Y
Y d Y
d Y d Y d
ef ef ef de
5.5 f
5
0 h 0.75 h 2 h 12 h 1 d 2d 3d 5d 7 d 10 d
Storage time
D ATP group Control group

6.5
X
a a a X
Y b Y
X
6 b c c Y X
X
X
c c X X X X X Y de
Y d de Y Y de Y de de
pH
d Y Y e Y e
fg ef ef ef ef
g
5.5
5
0 h 0.75 h 2 h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time
Fig. 10.5 The changes of pH in ground muscle at 25 C (a), 15 C (b), 4 C (c), and 1.5 C (d).
Different lowercase letters represent significant difference within the same group (P < 0.05). At the
same storage time, different capital case letters represent significant difference between ATP treated
group and the control group (P < 0.05)
Fig. 10.6 Gel images of and phosphorylation level of myofibrillar proteins. (a and b) Gel images of
myofibrillar phosphoproteins in ATP treated and the control group stained with Pro-Q Diamond;
(c and d), gel images of myofibrillar proteins in ATP treated group and the control group stained
with SYPRO Ruby. The phosphorylation level of myofibrillar proteins at 25 C (e), 15 C (f), 4 C
(g), and 1.5 C (h) in ATP treated and the control group. Different lowercase letters represent
significant difference within the same group (P < 0.05). At the same storage time, different capital
case letters represent significant difference between ATP treated group and the control group
(P < 0.05)
E
ATP group Control group
1.8

Xa Xa
Xb
Myofibrillar proteins
Ya Ya Yb Xc
1.2 Yc Xd
Yd
0.6
0
0h 0.75 h 2h 12 h 1d
Storage time
F ATP group Control group

1.8
Xa
Xb Xb
Ya Xbc Xc
Ya Ya Xd
1.2 Yb
Yb
Yc
0.6
0
0h 0.75 h 2h 12 h 1d 2d
Storage time
G ATP group Control group

1.8
Xa Xa Xa
Xb Xab Xa Xab
Xab
Xc Ya Yab Xc
Ybc Yb Ycd
1.2 Ye
Yde
Yf
Yg
Yh
0.6
0
0 h 0.75 h 2 h 12 h 1d 2d 3d 5d 7d 10 d
Storage time
H ATP group Control group
1.8
Xa
Xb
Xbc Xbc Xbc
c Xb Xbc Xbc
Xcd
e de
1.2 Yb Yb Yb Yb Yb
a Yb
Yc Yc
b b Yd
0.6
0
0 h 0.75 h 2 h 12 h 1d 2d 3d 5d 7d 10 d 14 d 21 d
Storage time

and 1.5 C were different compared with 25 C and 15 C. At 4 C, the P/T ratio
increased first and then decreased significantly (P < 0.05) after 3 days in the control
group, which indicated that the ATP content could maintain phosphorylation reac-
tion before 2 days and later, due to inadequate ATP content, phosphorylation
reaction was weakened and dephosphorylation was the dominated one. While in
ATP treated group at 4 C, the P/T ratio raised within 0.75 h (P < 0.05) and then kept
slightly changing with no significant difference (P > 0.05). The changes in phos-
phorylation level at 1.5 C were similar with 4 C because the phosphate groups
offered by ATP were sufficient to promote phosphorylation. The slower consump-
tion rate of ATP at low temperature made more phosphate group could be utilized in
postmortem muscle. The phosphorylation level of ATP treated group was higher
than the control group (P < 0.05) at each temperature, which testified that exogenous
ATP stimulated the elevation of phosphorylation level.
Besides the direct effect of phosphate group provided by ATP on phosphorylation
level, there was also indirect effect such as pH value impacted by ATP. Because
ATP was related to glycolysis, pH value in postmortem muscle mainly relied on
lactic acid produced by glycolysis. Huang et al. (2012) reported that the phosphor-
ylation level of myofibrillar protein was influenced by different pH decline rate at
early postmortem time, but had little effect on the extension of storage time. As the
result in Fig. 10.6, the phosphorylation level at early storage time of 25 C and 15 C
was higher than 4 C and 1.5 C, which was coherent with previous (Huang et al.
2012) conclusion that the phosphorylation level was higher in the group of higher
pH decline rate. Combined with the results in this study, the rate of ATP consump-
tion was a decisive factor of phosphorylation level in postmortem.
According to previous studies, the phosphorylated myofibrillar protein is princi-
pally involved in muscle contraction (Lametsch et al. 2011; Chen et al. 2016).
Muroya et al. (2007) reported that myosin regulatory light chain was phosphorylated
during rigor mortis formation. The increasing phosphorylation level of myosin light
chain 2, desmin, and actin was measured in aging muscle (Gannon et al. 2008).
Conclusively, ATP content might play an important role in muscle contraction and
meat tenderness through regulating myofibrillar protein phosphorylation.
10.3.4 Effect of ATP on Protein Degradation in Postmortem

Muscle
The degradation level of μ-calpain, desmin, and troponin T was measured by using
western blot and the result is shown in Fig. 10.7. With the increase of postmortem
time, μ-calpain was the most well-characterized type of calpain and showed more
proteolysis through autolysis (Huff-Lonergan et al. 1996). At 25 C in ATP treated
group, the bands of 80 kDa completely disappeared, showing that μ-calpain was
degraded to lower molecular weight (78–76 kDa) after 12 h of storage, while at 2 h in
the control group. Similarly, at 4 C, the degradation of 80 kDa was faster in the
226
10
Fig. 10.7 Degradation of μ-calpain (a, b), troponin T (c, d), and desmin (e, f) in ATP treated and the control groups at 25 C and 4 C
Effects of Temperature on Protein Phosphorylation
10.4 Effect of Temperature and pH on Dephosphorylation of Myofibrillar Protein In. . . 227
control group than the ATP treated group, which indicated the earlier autolysis and
activity of μ-calpain in control group. Therefore, the ATP content was negatively
related to degradation of μ-calpain and through increased phosphorylation level of
μ-calpain. However, the faster exhaustion of ATP in the control group led to the
early release of Ca2+, which attributed to the earlier autolysis of μ-calpain (Huang
et al. 2012). Desmin and troponin T, related to meat tenderness, were structural
proteins of the sarcomere and can be degraded by μ-calpain during postmortem
(Huff-Lonergan et al. 2010). Desmin was localized at the periphery of the myofi-
brillar Z-disk in skeletal muscle and degraded during postmortem storage (Huff-
Lonergan et al. 1996). Troponin T was strongly related to the shear force and
regulated the thin filament during muscle contraction through its interaction with
tropomyosin (Lehman et al. 2001). The bands of desmin in ATP treated group
(25 C) were degraded to lower molecular weight (approximately 38 kDa) at 12 h
of storage, and more rapid degradation occurred in the control group. The degrada-
tion trend of troponin T was similar to that of desmin. At 4 C, the degradation of
desmin and troponin T was quicker in the control group than the ATP treated group.
Therefore, the obviously slower degradation of desmin and troponin T was showed
in ATP treated group, where the autolysis of μ-calpain was inhibited as well. The
reason was that the phosphorylation of myofibrillar proteins reduced the suscepti-
bility of proteolytic degradation by μ-calpain (Li et al. 2017). Thus, ATP might
contribute to meat tenderness by affecting the phosphorylation and controlling
protein degradation.
10.4 Effect of Temperature and pH on Dephosphorylation

of Myofibrillar Protein In Vitro
This part mainly determined the effect of temperature (25 C, 15 C, 4 C, and
1.5 C) and pH (5.2, 5.8, and 6.4) on dephosphorylation of myofibrillar protein,
which would provide a theoretical basis in order to improve meat quality. Myofi-
brillar protein of postmortem muscle was extracted, and alkaline phosphatase
(AP) was added to catalyze dephosphorylation. The phosphorylation level was
detected by SDS-PAGE. Gels were stained by Pro-Q Diamond and SYPRO Ruby.
10.4.1 Effect of Temperature and pH on the Activity

of Alkaline Phosphatase
The optimal pH of AP is about 9–10, and the optimum temperature is about 40 C

(Wang and Yan 2009; Zhang and Guan 2012; Guo et al. 2004; Zhao et al. 2003). The
results of AP activity at different temperatures and pH are shown in Fig. 10.8. The
activity of AP gradually decreased with the extension of incubation time. At 25 C
Fig. 10.8 The AP activity (A) 3000 X X

of different temperatures YY a Y
a XX XX
a a Za Yb b XX
and pH. (a–d) Represent AP Y bc b Yc c
b c bc
activity of 25 C, 15 C, 2000
c
4 C, and 1.5 C.
King Unit/mL
AP activity
Different lowercase letters pH5.2
represent significant 1000 pH5.8
difference between different pH6.4
time points of the same
storage temperature 0
(P < 0.05). Different capital 10 min 30 min 1h 2h 4h
letters represent significant
Time
difference between different (B) 3000 X
Ya
storage temperatures at the a
XX
XX X
bb
same time point (P < 0.05). Z Y Y
c c
Zd
Yc
YY d
X
Translated from (Ren et al. a a

2000 b b b e
King Unit/mL
2019)
AP activity
pH5.2
1000 pH5.8
pH6.4
0
30 min 2h 4h 12 h 1d
Time
(C)
3000
XX
aa XX X X X
bb Y X
2000 Y Y Zc c ZY c YY c YY d
King Unit/mL
d a de
b ab ab ab ab e
AP activity
pH5.2
1000 pH5.8
pH6.4
0
30 min 4 h 12 h 1 d 2d 4d
Time
D
700
XX X
XX
Y a ab Y Y a Y b bc
a ab a bc d
cd
King Unit/mL
a a cd add
AP activity
pH5.2
350
pH5.8
pH6.4
0
30 min 12 h 1d 2d 4d 7d
Time
during 1–4 h, AP activity was 2115.27–2258.25 King unit/mL at pH 5.8 and 6.4,
which was significantly higher than 1912.53–2063.10 King unit/mL at pH 5.2
(P < 0.05). The result suggested that pH 5.2 restrained AP activity at 25 C

(P > 0.05).
When incubating for 30 min, 12 h, 1 day at 15 C, the AP activity at pH 6.4 was
significantly higher than pH 5.2 and pH 5.8 (P < 0.05). This result showed that when
temperature decreased, the lower the pH, the stronger the inhibitory effect on AP
activity. At 4 C and pH 5.2, the highest AP activity was 1805.22 King unit/mL at
2 days. It showed that as the incubation time prolonged, the inhibition of lower
temperature and pH would decrease.

Level of Myofibrillar Protein
Myofibrillar protein was stained by Pro-Q Diamond and SYPRO Ruby after
SDS-PAGE (Fig. 10.9). Protein dephosphorylation can be catalyzed by phosphatase
and reduce protein phosphorylation level (Silverman-Gavrila et al. 2009; Hunter
1995). As shown in Fig. 10.10, with the extension of incubation time, there was no
significant difference in phosphorylation level of the control group at different
temperatures and pH (P > 0.05). The result suggested that myofibrillar protein
could not be dephosphorylated without AP. For pH 6.4, when incubating for 4 h
at 25 C, myofibrillar protein phosphorylation level of AP treated group was
significantly lower than that of the control group (P < 0.95 0.05); at 15 C,
myofibrillar protein phosphorylation level of AP treated group was significantly
lower than that of the control group (P < 0.05); at 4 C and 4 days, myofibrillar
protein phosphorylation level of AP treated group was significantly lower than that
of the control group (P < 0.05). For pH 5.8, when incubating for 4 h, 1 day, 4 days at
25 C, 15 C, 4 C, myofibrillar protein phosphorylation level of AP treated group
was significantly lower than that of the control group (P < 0.05). These findings
suggested high temperature was advantageous for AP catalyzing dephosphorylation
of myofibrillar protein. For pH 5.2, myofibrillar protein phosphorylation level of AP
treated group and the control group had no significant difference (P > 0.05), which
showed that AP activity was inhibited at pH 5.2.
In conclusion, AP catalyzed the dephosphorylation of myofibrillar protein, which
was regulated by both temperature and pH value. However, when a factor deviated
from the optimal condition, it would inhibit AP catalyze dephosphorylation.

Level of AP
The molecular weight of AP used in this experiment was 140–160 kDa. AP was a
homologous dimer phosphomonoesterase (Bi 2015), which would be denatured
Fig. 10.9 Gel images of Pro-Q and Ruby stained by SDS-PAGE of different temperatures and pH
values. AP means AP treated group, C means the control group, M means marker, S means
standard. A1, B1, C1, D1: Pro-Q stained gel images of 25 C, 15 C, 4 C, 1.5 C; A2, B2, C2,
D2: Ruby stained gel images of 25 C, 15 C, 4 C, 1.5 C. Translated from (Ren et al. 2019)
when preparing for electrophoresis samples. After the disassembly of the dimer, the
molecular weight was about 70 kDa, as shown in Fig. 10.11. The alkaline phospha-
tase phosphorylation level changed significantly during incubation, as shown in
Fig. 10.10. With the extension of incubation time, the phosphorylation level of AP
was significantly changed. At different temperatures and pH, the phosphorylation
level of AP was gradually decreased, indicating that AP could not only catalyze
myofibrillar dephosphorylation but also catalyze dephosphorylation of itself. At
25 C, AP phosphorylation level was significantly different (P < 0.05) when
incubating for 10 min; at 15 C, AP phosphorylation level was significantly different
(P < 0.05) when incubating for 30 min; at 4 C, AP phosphorylation level was
significantly different (P < 0.05) after 4 h incubation. This result suggested that
rising temperature would move up the time of dephosphorylation. At later stage of
incubation, AP phosphorylation level of pH 5.2 was significantly higher than that of
other two pH levels (P < 0.05), indicating that pH 5.2 had a stronger inhibition effect
on AP, even though the temperature increased. When AP was dephosphorylated, its
activity would decrease slightly, the main factors affecting AP activity were tem-
perature and pH.
Therefore, postmortem storage temperature affected protein phosphorylation and
meat quality development at least by affecting pH decline and ATP consumption in
postmortem muscle.
(A) 1.5
a
a a *
a b ab
ab b ab ab b *
ab AP5.2
1 b b
P/T ratio
c C5.2
AP5.8
0.5 C5.8
AP6.4
C6.4
0
10 min 30 min 1h 2h 4h
(B) 1.5 Time
* * * * *
a a ab *
a ab a a AP5.2
1 b c c
P/T ratio
C5.2
AP5.8
0.5 C5.8
AP6.4
C6.4
0
30 min 2h 4h 12 h 1d
Time
(C) 1.5
* * * *
a a a ab a ab
ab a ab a b
b AP5.2
1
P/T ratio
C5.2
AP5.8
0.5 C5.8
AP6.4
C6.4
0
30 min 4h 12 h 1d 2d 4d
Time
D)
1.5
AP5.2
1
P/T ratio
C5.2
AP5.8
C5.8
0.5
AP6.4
C6.4
0
30 min 12 h 1d 2d 4d 7d
Time
Fig. 10.10 Myofibrillar protein phosphorylation level of different temperatures and pH. (a–d)
Represent AP activity of 25 C, 15 C, 4 C, and 1.5 C. AP means AP treated group, (c) means
the control group. Different lowercase letters represent significant difference between different time
points of the same pH (P < 0.05). * represents significant difference between different group at the
same time point (P < 0.05). Translated from (Ren et al. 2019)
A B
1.5 1.5 X
XXY XX X
a X X
X
a a Y ab a ab Y
X
a Y a X
P/T ratio of AP
a b a Y Y
P/T ratio of AP
Y b b b X
1 b 1 a Z c
pH5.2 b
b pH5.2
X
Z
c Y
c pH5.8 pH5.8
Z Y Z
0.5 c Y
d c Z pH6.4 0.5 c
d Z YY pH6.4
d d e d
0 0
10 min 30 min 1h 2h 4h 30 min 2h 4h 12 h 24 h
Time Time
C
1.5 (D)
XX 2
a
a a ab XX
a Y bb X
X
P/T tatio of AP
b X X d
1 cX c a a
c c X pH5.2 1.5 a X X
P/T ratio of AP
a
d a ab b a a c cX
pH5.8 b
a
1 pH5.2
0.5 pH6.4
Y Y
Z YY Y pH5.8
d d dd c
d Y pH6.4
Y Y
0.5 b b b
0
30 min 4h 12 h 1d 2d 4d
0
Time
30 min 12 h 1d 2d 4d 7d
Time
Fig. 10.11 AP phosphorylation level of different temperatures and pH values. A, B, C, and D

represent AP phosphorylation level of 25 C, 15 C, 4 C, and 1.5 C. Different lowercase letters
represent significant difference between different time points of the same pH (P < 0.05). Different
capital letters represent significant difference between different pH at the same time point
(P < 0.05). Translated from (Ren et al. 2019)
10.5 Conclusions
Temperature affected phosphorylation and dephosphorylation through pH changes

and ATP content. The proteins involved in glycolysis and muscle construction were
in two clusters. Through correlation analysis, ATP was a more important regarding
influencing the phosphorylation level of individual protein. The higher ATP content
was positively related to phosphorylation level of myofibrillar proteins and
restrained the degradation of μ-calpain, troponin T, and desmin. The activity
of alkaline phosphatase was lower at 1.5 C and pH 5.2, so that dephosphorylation
of myofibrillar proteins was not significant. The study gave a clear understanding of
phosphorylation and dephosphorylation in postmortem muscle, as well as the rela-
tionship between ATP content and protein degradation.
Agriculture, 100, Ren, C., et al., Effects of temperature on protein phosphorylation in postmortem
muscle, 551–559. Copyright (2020), with permission from Elsevier. Parts of this chapter are
translated from Food Science, 40(16), REN, C., et al., Effect of temperature and pH on dephos-
phorylation of myofibrillar protein in vitro (Chinese), 1–7. Copyright (2020), with permission from
Journal of Food Science.
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Chapter 11
Effects of Ionic Strength on Protein
Phosphorylation
Abstract Salting is one of the most ancient preservation methods for food, which is
widely used to preserve meat and improve the quality of meat products, such as
water holding capacity (WHC), texture, tenderness, flavor, taste, and so on. Protein
phosphorylation is one of the most ubiquitous post-translational modifications
(PTMs). Reversible protein phosphorylation plays an important role in protein
structure, function, signaling, and regulation. As one of the most essential processes
for meat products, the effects of salting on muscle protein phosphorylation are
unclear. Thus, we investigated the phosphorylation of myofibrillar and sarcoplasmic
proteins in response to salt content and salting time. We discovered that salting
showed significant effect on myofibrillar protein phosphorylation. Four categories of
phosphorylated protein were identified by liquid chromatography-tandem mass
spectrometry (LC-MS/MS), involved in stress response (heat shock protein),
glycometabolism (glycogen phosphorylase, glyceraldehyde-3-phosphate dehydro-
genase), oxidation or reduction (superoxide dismutase), and others (myoglobin). The
phosphorylation level of the proteins mentioned above was affected by salting. Thus,
salting might influence meat quality through protein phosphorylation, which regu-
lated protein degradation and glycolysis. Furthermore, ten different phosphoproteins
(>1.5 fold) induced by salting were identified by LC-MS/MS and the UniProt
database, most of which were involved in glycometabolism, protein function, and
protein degradation. It was concluded that salting might influence meat quality
through protein phosphorylation, which regulated glycolysis metabolism, protein
function, and degradation.
Keywords Protein phosphorylation · Salting · Protein degradation · Glycolysis ·

Differential phosphorylated proteins · Salting temperature · Sodium chloride · Meat
quality

https://doi.org/10.1007/978-981-15-9441-0_11
238 11 Effects of Ionic Strength on Protein Phosphorylation
11.1 Introduction
Salting is commonly used in meat processing to improve the quality of processed

meat products, such as flavor, water holding capacity, tenderness, juiciness, firm-
ness, gelation, and appearance (Comaposada et al. 2000; Duranton et al. 2012; Shao
et al. 2016). NaCl is the essential salting material used in cured or cooked meat
products. It increases the ionic strength within muscle, which causes protein disso-
ciation, denaturation, modification, and myofibril swelling (Kim et al. 2015; Shao
et al. 2016; Wang et al. 2016).
Protein phosphorylation is the most common post-translational modification
(PTM). The majority of muscle proteins can be phosphorylated in vivo. Many
salt-responsive phosphoproteins also have been identified in vivo in plants
(Hu et al. 2013). In mice, salt regulates the phosphorylation of STE20/SPS1-related
proline/alanine-rich kinase (SPAK) and Na-K-Cl cotransporter isoform 1 (NKCC1)
in aorta (Zeniya et al. 2013). Higher salt intake decreases the phosphorylation of
with-no-lysine kinase (WNK)-SPAK-NKC1 in the vascular smooth muscle,
resulting in disorganized common vascular tone (Zeniya et al. 2013). Protein
phosphorylation affects protein stability, enzymatic activity, and muscle contraction,
which may then affect muscle development, carcass rigor mortis, and meat quality
(Huang et al. 2012; Li et al. 2012). NaCl influences meat quality by regulating
myofibrillar protein denaturation, actomyosin dissociation, myosin hydration, and
myoglobin redox (Duranton et al. 2012; Kim et al. 2015; Wang et al. 2016), while
myosin, actin, and myoglobin could be phosphorylated (Li et al. 2017; Chen et al.
2016).
The standard curing temperature in modern meat production is generally 4 C.
However, in actual production, a few small enterprises adopt room temperature for
pickling (25 C). In addition, ice temperature preservation technology between 0 C
and freezing point temperature has become a research hotspot in recent years. With
the application of ice temperature preservation technology, ice temperature
curing also becomes a research direction. Garcia-Gil et al. (2014) studied the effect
of curing temperature on salt absorption of ham and found out that low-temperature
curing was not conducive to salt absorption (Garcia-Gil et al. 2014). Protein phos-
phorylation was affected by many factors, the endogenous factors were mainly the
action of protein kinases and phosphatases (Rubin and Rosen 1975), while the
exogenous factors included factors that affected enzymes. Besides, temperature
was a very important factor affecting enzyme activity.
Whether NaCl concentration and salting temperature influence protein phosphor-
ylation, and what effects of salting on muscle protein phosphorylation should be
elucidated by further research. In this case, systematically studying the response of
different phosphorylated proteins to sodium chloride will not only help us under-
stand the relations of the salting and muscle protein phosphorylation, but also help
reveal a potential new mechanism of salting changing meat quality.
11.2 Phosphorylation Level and Influencing Pathway of Myofibrillar and Sarcoplasmic. . . 239
11.2 Phosphorylation Level and Influencing Pathway

of Myofibrillar and Sarcoplasmic Proteins of Muscle
in Response to Salting
Phosphorylation level of myofibrillar and sarcoplasmic proteins of muscle in

response to salting was discovered recently. In order to explore it clearly, the
myofibrillar and sarcoplasmic proteins were extracted from salted meat with 0, 1,
2, 3, 4, and 5% salt and salted for 0, 2, 4, 6, 8, and 16 h. Phosphorylation level was
analyzed by dodecyl sulfate sodium salt and polyacrylamide gel electrophoresis
(SDS-PAGE), fluorescence staining, and LC-MS/MS. Four categories of phosphor-
ylated protein were identified, which were involved in stress response (heat shock
protein), glycometabolism (glycogen phosphorylase, glyceraldehyde-3-phosphate
dehydrogenase), oxidation or reduction (superoxide dismutase), and others (myo-
globin). Briefly, salting might influence meat quality through protein phosphoryla-
tion, which regulated protein degradation and glycolysis (Zhang et al. 2016a).
11.2.1 Global Phosphorylation of Myofibrillar Proteins

with Salting Time
The amount of salt added in traditional meat processing is typically 2% (Mora-

Gallego et al. 2016). Therefore, the 2% salt was chosen as the experimental dose to
profile protein phosphorylation during meat salting. As shown in Fig. 11.1a and b, a
total of 23 bands of myofibrillar proteins were detected by Quantity One and selected
for the analysis. The global phosphorylation level of proteins was shown in
Fig. 11.1c. The phosphorylated proteins divided by total proteins (P/T ratio) of
each sample were evaluated by the ratio of the intensity of all bands in Pro-Q
Diamond images to the intensity of all bands in SYPRO Ruby images. The P/T
ratio of control group increased from 0 to 2 h, then decreased slightly with no
significant difference during the whole salting process (P > 0.05). Conversely, the
P/T ratio of the salted meat with 2% salt significantly decreased from 0 to 16 h
(P < 0.05).
The results showed that global phosphorylation level of myofibrillar proteins was
significantly decreased (P < 0.05) during the salting development after adding 2%
salt, while it had no significant difference in the control group. The global phos-
phorylation level of salted group was lower than the control group, which indicated
that salt affected protein phosphorylation. The global phosphorylation level at 16 h
A B
0% salt 2% salt 0% salt 2% salt
M 0h 2h 4h 6h 8h 16h 2h 4h 6h 8h 16h M 0h 2h 4h 6h 8h 16h 2h 4h 6h 8h16h
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11
12 11
13 12
14 13
15 14
16 15
17 16
18 17
18
19
20 19
20
21
22 21
22
23
23
C
0.9
0% salt
2% salt
0.8
P/T ratio
a
a
0.7 a ab
0.6 b
c
0.5
0h 2h 4h 6h 8h 16h
salting time (h)
Fig. 11.1 Phosphorylation of myofibrillar proteins during salting. (a–b) SDS-PAGE analysis of
phosphorylated proteins by Pro-Q Diamond staining (a) and total myofibrillar proteins by SYPRO
Ruby staining (b). (c) Quantification of global protein phosphorylation level of myofibrillar proteins
at different time points during salting. Different letters indicate significant difference with salting
time (P < 0.05). Asterisk (*) shows significant differences between the 0% salt and 2% salt groups
in the same salting time (P < 0.05) (Zhang et al. 2016a)
was lowest during the salting development, so we further studied the influence of salt
on muscle protein phosphorylation by adding different concentration of salt.
The phosphorylation level of myofibrillar proteins with different salt concentra-
tion was shown in Fig. 11.2. Compared to control group, the phosphorylation of
myofibrillar protein of meat salted with 2~5% (m/v) NaCl for 16 h was reduced.
Meat salted by 3% salt had the lowest protein phosphorylation (Fig. 11.2c), showing
that salt influenced protein phosphorylation during meat processing.
P/T ratio of band 8, 16, 19, and 23 was greater than 1, so these proteins were
highly phosphorylated. The P/T ratio of individual protein bands was shown in
Fig. 11.2d. In general, the protein phosphorylation level of most bands from salted
meat was lower than that of the control group except for band 16 and 20, which
showed an increase in protein phosphorylation after salting.
By investigation, it was discovered that salting decreased phosphorylation of
myofibrillar proteins. Myofibrillar proteins were structural proteins responsible for
sarcomere structure integrity and muscle contraction, both of which were related to
meat tenderness. Salting improved meat tenderness (Sheard and Tali 2004), while
many researchers found out that protein phosphorylation may affect meat quality
(Huang et al. 2012; Li et al. 2015; Chen et al. 2016). Our study showed that the
global phosphorylation level of myofibrillar proteins in salted meat was reduced.
This was in consistence with previous literature, which reported that protein phos-
phorylation affected meat tenderness and tender meat had lower protein phosphor-
ylation level (Chen et al. 2016; Zolla 2012). The phosphorylation of myofibrillar
proteins reduced the degradation of these proteins by calpains, such as actin binding
protein, Troponin T (TnT), and Troponin I (Di Lisa et al. 1995; Toyo-Oka 1982;
Zhang et al. 1988). In this study, the phosphorylation of myosin heavy chain (band
3), myosin binding protein C (band 4), and actin (band 11) was reduced by salting,
while these bands were related to meat tenderness. According to all the studies, we
speculated that relatively low phosphorylation level of myofibrillar proteins in salted
meat made them more easily to be degraded than the control group, as a result,
improved meat tenderness.
Rigor mortis or muscle contraction shortens sarcomere length and decreases meat
tenderness. Many phosphorylated proteins identified in muscle are related to sarco-
mere function which affects meat quality, so it is speculated that protein phosphor-
ylation may be related to muscle contraction and quality change (Huang et al. 2011).
The phosphorylation of myosin light chain (MLC) enhances the intensity of con-
traction (Stull et al. 2011). Our result showed that the phosphorylation of MLC
(Fig. 11.2d, band 23) was reduced by salting, which might inhibit muscle contraction
and further affect meat tenderness, providing another mechanism that salting could
increase meat tenderness.
A B
M 0% 1% 2% 3% 4% 5% M 0% 1% 2% 3% 4%
1 5% 1
2 2
3 3
4 4
5 56
6
7 7
8 8
9 9
10 10
11 11
12 12
13 13
14 14
15 15
16 16
17
18 17
18
19 19
20 20
21 21
22 22
23 23
C
1
a ab bc cd c
0.8
c
0.6
P/T ratio
0.4
0.2
0
0% 1% 2% 3% 4% 5%
salt content ( %)
Fig. 11.2 Phosphorylation of myofibrillar proteins in response to salt on concentrations. (a–b)

SDS-PAGE analysis of phosphorylated proteins by Pro-Q Diamond staining (a) and total myofi-
brillar proteins by SYPRO Ruby staining (b). (c) Quantification of global protein phosphorylation
level of myofibrillar proteins from meat salted at different salt concentrations. (d) Protein phos-
phorylation level of nine individual protein bands. Different letters indicate significant differences
(P < 0.05) (Zhang et al. 2016b)
D
1 1.8
Band 3 Band 4 Band 8
0.8 1.5
0.3
0.6 1.2
P/T ratio
P/T ratio
0.2 0.9
P/T ratio
0.4
0.6
0.1
0.2 0.3
0.0 0 0.0
4%
0%
1%
2%
3%
5%
0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5%
Band 10 0.25 Band 11 1.5 Band 16

0.8 0.20
1.0
P/T ratio
P/T ratio
P/T ratio
0.6 0.15
0.4 0.10
0.5
0.2 0.05
0.0 0.00 0.0
0% 1% 2% 3% 4% 5% 0%1%2%3%4%5% 0%1%2%3%4%5%
2.0 0.8
P/T ratio
Band 19 Band 20 8
Band 23
1.5 0.6 6
P/T ratio
P/T ratio
1.0 0.4 4
0.5 0.2 2
0.0 0.0 0
0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5% 0%1%2%3%4%5%
11.2.2 Global Phosphorylation of Sarcoplasmic Proteins

with Salting Time
In total, 20 sarcoplasmic protein bands were detected to be phosphorylated in meat

(Fig. 11.3a). Unlike to myofibrillar proteins, salting showed no significant effect on
the phosphorylation of sarcoplasmic proteins. The P/T ratios of both the control
group and salted meat with 2% salt showed no significant difference (P > 0.05) at
different salting time points, though an increasing trend in P/T ratio with salting time
might exist in the salted meat (Fig. 11.3c).
To confirm that salt had no effect on the global phosphorylation level of sarco-
plasmic proteins, sarcoplasmic proteins were extracted from meat and salted for 16 h
with different concentrations of salt. SDS-PAGE and fluorescence staining with
A B
M 0h 2h 4h 6h 8h16h 2h 4h 6h8h16h M 0h 2h 4h 6h 8h 16h2h 4h 6h 8h16h
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11 11
12 12
13 13
14 14
15 15
16 16
17 17
18 18
19 19
20 20
21
C 0.60
0% salt
0.55 2% salt
P/T ratio
0.50
0.45
0.40
0.35
0h 2h 4h 6h 8h 16h
salting time (h)
Fig. 11.3 Phosphorylation of sarcoplasmic proteins during salting (a–b) SDS-PAGE analysis of
phosphorylated proteins by Pro-Q Diamond staining (a) and total sarcoplasmic proteins by SYPRO
Ruby staining (b). (c) Quantification of global protein phosphorylation level of sarcoplasmic
proteins at different time points during salting (Zhang et al. 2016a)
Pro-Q Diamond dye were conducted to detect phosphoproteins. As shown in

Fig. 11.4a–c, no difference was determined (P > 0.05) in the P/T ratios among
A B
M 0h 2h 4h 6h 8h 16h 2h 4h 6h 8h 16h M 0h 2h 4h 6h 8h 16h 2h 4h 6h 8h

C
0.6
0.5
0.4
P/T ratio
0.3
0.2
0.1
0
0% 1% 2% 3% 4% 5%
salt content (%)
Fig. 11.4 Phosphorylation of sarcoplasmic proteins in response to salt concentrations. (a–b)

SDS-PAGE analysis of phosphorylated proteins by Pro-Q Diamond staining (a) and total sarco-
plasmic proteins by SYPRO Ruby staining (b). (c) Quantification of global protein phosphorylation
level of sarcoplasmic proteins from meat salted at different salt concentrations. (d) Protein phos-
phorylation level of 15 individual protein bands (Zhang et al. 2016b)
D
1.5 Band 1 1.5
0.8 Band 5
Band 3
1.2
0.6
1.0
P/T ratio
0.9
P/T ratio
P/T ratio
0.4
0.6
0.5
0.3 0.2
0.0 0.0 0.0

0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5%
0.2 1.0 Band 8

Band 7
3.0 Band 6 0.8
P/T ratio
2.0 0.6
P/T ratio
0.1
0.4
P/T ratio
1.0 0.2
0.0 0.0 0.0

0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5%
1.0 0.3 0.8 Band 12

Band 10 Band 11
0.6
0.2
P/T ratio
0.4
P/T ratio
0.5
P/T ratio
0.1 0.2
0.0 0.0 0.0
0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5%
0.20 1.5 Band 14 0.15 Band 16

Band 13
1.2 0.12
P/T ratio
0.9 0.09
P/T ratio
P/T ratio
0.10
0.6 0.06
0.3 0.03
0.00 0.0 0.00
0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5%
Band 21
1.2 Band 17 8 Band 19 0.25
6 0.20
0.9
P/T ratio
0.15
P/T ratio
P/T ratio
0.6 4
0.10
0.3 2 0.05
0.0 0 0.00
0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5% 0% 1% 2% 3% 4% 5%

different treatments, confirming that salt had no significant effect on the global
phosphorylation level of sarcoplasmic proteins of meat. However, the phosphoryla-
tion in most protein bands was affected by salt (Fig. 11.4d). The phosphorylation
level of eight protein bands was decreased by salt, whereas that of five bands was
increased by salt. Although salt had no significant effect on the global phosphory-
lation of sarcoplasmic proteins, it affected the phosphorylation of individual pro-
teins. The results indicated that salt also had effect on the phosphorylation of
sarcoplasmic proteins.
The identified sarcoplasmic proteins by LC-MS/MS are highly abundant. The P/T
ratio is a semi-quantitative method for protein phosphorylation (Huang et al. 2011;
Schulenberg et al. 2003; Silverman-Gavrila et al. 2009). Several distinctive phos-
phorylated bands of sarcoplasmic proteins were selected to be identified by LC-MS/
MS. The functional categories of the identified proteins were matched manually on
the basis of the retrieved information in the Protein Knowledge-base (UniProtKB in
www.uniprot.org). According to the score, the theoretical molecular weight, number
of matched peptides, and the coverage of the entire amino acid sequence (Chen et al.
2016), proteins were identified from each gel band (Table 11.1). In total, four
categories of proteins were identified from these bands, including stress response,
glycometabolism, oxidation or reduction, and others. Stress response proteins
included heat shock protein alpha (HSPα) and heat shock protein 70 (HSP70).
Glycometabolism proteins included glycogen phosphorylase (GP), glyceralde-
hyde-3-phosphate dehydrogenase (GAPDH), enolase (ENO), AMP-activated pro-
tein kinase (AMPK), aldolase (ALD), and phosphoglycerate kinase (PGK).
Oxidation or reduction proteins included superoxide dismutase (SOD) and aldehyde
dehydrogenase (ALDH). Other proteins included 14-3-3 protein and myoglobin
(Mb).
11.2.3 Salting Influences the Phosphorylation of Glycolytic

Enzymes
Glycolysis, which is regulated by glycolytic enzymes, is the principal way for energy
supply in postmortem muscle and related to meat quality (Donnelly and Finlay
2015). The phosphorylation of many glycolytic enzymes or regulatory proteins
(GP, GAPDH, ENO, AMPK, ALD, PGK) has been identified to be influenced by
salting. Phosphorylation of glycolytic enzymes affected their activity and stability
(Huang et al. 2011; Sale et al. 1987; Müller et al. 2015). GP was an enzyme involved
in glycolysis reaction. It was activated into active enzyme form by phosphorylation,
catalyzed by phosphorylase kinase, and then further promoted glycolysis (Müller
et al. 2015). The phosphorylation of band 5 (GP) in 3% salt group was higher than
other groups, so GP had higher activity, promoted glycolysis and then regulated
meat tenderness. GAPDH had many post-translation modifications. Phosphorylation
of GAPDH decreased its activity and then inhibited the glycolysis (Bell and Storey
Table 11.1 Identification of phosphorylated sarcoplasmic proteins by LC-MS/MS. Adapted from (Zhang et al. 2016b)
248
Band Accession MW Seq.

Protein namesa no.b noa Speciesc (Da)d Scoree MPf Cov.g Function
Acetyl-CoA carboxylase 1 1 Q28559 OS 266,736 67 3 2% Biotin enzyme
Uncharacterized protein 3 F1MHT1 BOS 176,199 19,863 769 69%
Glycogen phosphorylase 5 O18751 OS 97,702 44,666 1713 77% Glucose
metabolism
Hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coen- 6 B6UV59 OS 83,926 2687 79 50% Coenzyme Binding
zyme A thiolase/enoyl-coenzyme A hydratase (Trifunctional
protein) alpha subunit
Heat shock protein alpha 6 A8DR93 OS 85,077 1181 37 34% Stress response
Heat shock protein 70 7 H2DGR2 OS 70,536 5377 207 58% Stress response
11
Heat shock protein 70 7 H2DGR0 OS 70,471 5208 199 49% Stress response
HSP70 7 H9LHX6 OS 69,673 1130 38 8% Stress response
Malic enzyme 8 B5SZL5 OS 64,601 705 23 61% Glucose
metabolism
AMP-activated protein kinase alpha2 subunit 8 A8D245 OS 62,833 50 1 9% Glycolysis
Protein disulfide isomerase family A member 3 10 C6JUQ0 OS 57,379 546 24 39% Rearrangement of
-S-S- bonds
Aldehyde dehydrogenase 1 family member L1 (fragment) 10 I1U3B9 OS 56,692 242 9 3% Oxidation
reduction
Enolase (fragment) 11 Q9N113 OS 11,952 14,465 427 81% Glycolysis
Phosphoglycerate kinase 12 B7TJ13 OS 44,936 13,304 446 73% Glycolysis
Beta-actin variant 2 12 D7RIF5 OS 42,052 2113 109 51% Sarcomeric
function
Enolase (fragment) 13 Q9N113 OS 11,952 1974 50 79% Glycolysis
Aldolase A (fragment) 13 Q9N116 OS 15,432 1900 58 61% Glycolysis
Aldolase A (fragment) 14 Q9N116 OS 17,291 522 9 74% Glycolysis
Glyceraldehyde-3-phosphate dehydrogenase 15 D7R7V6 OS 36,110 62,155 2031 78% Glycolysis
Effects of Ionic Strength on Protein Phosphorylation
Lactate dehydrogenase A (fragment) 16 Q9N109 OS 13,391 15,092 534 73% Glycolysis
11.2
14-3-3 protein epsilon 17 P62262 OS 29,326 4074 140 64% Cell apoptosis
Superoxide dismutase 18 C8BKD6 OS 24,736 2769 104 69% Oxidation
reduction
RAN 18 C5IJA0 OS 24,579 1113 46 61% GTP binding
Cytochrome c oxidase subunit 2 18 B0EXQ6 OS 26,134 627 23 23% Oxidation
reduction
AK1 19 C5IJA8 OS 21,750 14,379 623 82% Creatine energy
metabolism
Peroxiredoxin 2 19 C8BKC5 OS 22,059 1768 76 69% Cell redox
homeostasis
Alpha-crystallin B chain 20 Q5ENY9 OS 20,054 6559 341 48% Aggregation of
proteins
S-phase kinase-associated protein 1A 20 B8QGA5 OS 18,817 257 7 49% Kinase activity
Myoglobin 21 P02190 OS 17,044 32,908 962 97% Oxygen transport
a
Protein names and accession numbers were obtained from the UniProt database
b
Band numbers of the identified proteins
c
OS ovis aries, BOS bovine aries
d
Molecular mass of identified proteins (recorded in the UniProt database)
e
The MASCOT baseline significant score is 70. For the proteins identified in more than one band, the highest score was presented
f
g
Phosphorylation Level and Influencing Pathway of Myofibrillar and Sarcoplasmic. . . 249
2014). Finally, although protein oxidation was not analyzed, the influence of salt on
the phosphorylation status of SOD, peroxidase, and ALDH suggested that salting
might also impact protein oxidation and thus meat quality.
11.2.4 Salting Regulates the Phosphorylation of Heat Shock

Proteins
Heat shock proteins (HSPs) were identified in the phosphorylation proteins by

LC/MS/MS, such as HSPα and HSP70. HSPs were stress response proteins, and
they were divided into multiple subfamilies and had several post-translational
modifications which altered their functional roles. HSPα and HSP70 were essential
proteins in protection against stress. HSP70 in the myocardia, liver, and soleus
muscles was prone to phosphorylation after exercise (González and Manso 2004;
Melling et al. 2008). In this study, the phosphorylation of HSPα and HSP70 was
detected to be reduced by salting, which may alter their functional roles. Since HSPs
had impact on meat tenderness (Carvalho et al. 2014), our study further revealed that
salting might alter meat tenderness by regulating the phosphorylation of HSPs.
11.2.5 Research Prospect
In summary, salt influenced phosphorylation and dephosphorylation of muscle pro-

teins during salting. Salt decreased the global phosphorylation level of myofibrillar
proteins and affected the phosphorylation level of individual sarcoplasmic proteins.
The study may provide a new insight into the mechanism by which salting influences
the quality of processed meat products, especially the meat tenderness which is
related to protein degradation and muscle contraction. In future, the influencing
pathway of salt on myofibrillar and sarcoplasmic protein phosphorylation should be
studied more clearly in vivo or in vitro, thus making it possible to explain the protein
structure and dynamic function systematically.
11.3 Identification of Specific Phosphorylated Proteins

Induced by Ionic Strength
Salting may influence meat quality through protein phosphorylation, which regulates
glycolysis metabolism, protein function, and degradation. In order to explore it, ten
topside muscles of crossbred sheep were ground, mixed, and divided into two
groups, which were cured for 16 h with 0 and 3% NaCl, respectively. Muscle
proteins of two cured groups were analyzed by two-dimensional electrophoresis
11.3 Identification of Specific Phosphorylated Proteins Induced by Ionic Strength 251
coupled with Pro-Q Diamond and SYPRO Ruby staining. The different phosphor-
ylated proteins were determined using LC-MS/MS and the UniProt database. Ten
different phosphoproteins (>1.5 fold) induced by salting were identified, including
triosephosphate isomerase, glycogen phosphorylase, creatine kinase M-type, myo-
globin, troponin T fast skeletal muscle type, actin, myosin light chain 1/3, tropomy-
osin beta chain, etc. Most of the different phosphoproteins were involved in
glycometabolism, protein function, and protein degradation (Wang et al. 2017).
11.3.1 Identification of Differentially Phosphorylated

Proteins After Salting
After electrophoresis, gels were stained with Pro-Q Diamond for phosphoproteins
(P) and with SYPRO Ruby for total proteins (T). The relative phosphorylation level
of proteins was evaluated by the ratio of relative protein abundance between control
and salted group. As shown in Fig. 11.5, 13 protein spots were detected to be
differentially phosphorylated (ratio of relative protein abundance <2/3 or >1.5).
The isoelectric point (pI) of the differentially phosphorylated proteins was pH 5–8
and the molecular weight was 10–100 kDa. The proteins within the 13 spots were
identified by LC-MS/MS, including myoglobin (1, MB), myosin light chain 1/3-
skeletal muscle isoform (2, 20, 81, MYL1), tropomyosin beta chain (10, TPM2),
actin (18, 62, 84), troponin T fast skeletal muscle type (56, 70, TNT), apolipoprotein
A-I preproprotein (124, APOA1), triosephosphate isomerase (125, TPI) and F-actin-
capping protein subunit alpha-2 (142, CAPZA2). The relative abundance of phos-
phoproteins within the 13 spots was quantified by densitometric analysis (Fig. 11.6).
The phosphorylated values of spot 2, 81, 84, 124, 125, and 152 in control group were
higher than in salt group, and the values of other spots were lower in control group.
The two-dimensional electrophoresis (2-DE) gel images with SYPRO Ruby
staining of control group and salt group were showed in Fig. 11.7. Four differential
protein spots (>1.5 fold) on the SYPRO Ruby staining gels were excised out with
pipette tip, trypsin digestion and LC-MS/MS analysis were conducted. As a result,
creatine kinase- M type (95, 100, CK), troponin T fast skeletal muscle type
(97, TNT), and glycogen phosphorylase (293, GP) were identified. The gray level
value of the four differential proteins was presented in Fig. 11.8.The gray level of
spots 97 and 293 was not differential (1.5 > ratio of relative protein abundance >2/
3) in Pro-Q Diamond staining (P), but difference was much higher in salt group (ratio
of relative protein abundance <2/3 or >1.5) subjected to SYPRO Ruby staining (T),
resulting in higher phosphorylation level (P/T) of CK (97) and GP (293) in salt group
than that in the control group. Similarly, the value of spot 95 and 100 in control
group was lower than the salt group. Conclusively, the 17 spots were identified to be
10 kinds of protein (Tables 11.2 and 11.3), which were classified to glycolysis
metabolism enzymes (GP, CK, TPI), function proteins (myoglobin, APOA1), and
muscle contractile relative proteins (MYL1, TPM2, actin, TNT, CAPZA2).
A pI 3-10 B pI 3-10
84
142 84 81 142 81
62
SDS-PAGE
62
10 10
18 70 18 70
56 56
124 20 124 125

20 125 1 1
2 2
Fig. 11.5 Images of gels stained with Pro-Q Diamond to detect phosphoproteins. (a) control;
(b) meat salted with 3% NaCl (Wang et al. 2017)
3
control group
2.5 salt group
Relative protein abundance
1.5
0.5
0
1 2 10 18 20 56 62 70 81 84 124 125 142
The number of protein spots
Fig. 11.6 Quantification of phosphoproteins stained by Pro-Q Diamond in selected spots which
were different between control and salted meat in protein abundance (the ratio <2/3 or >1.5) (Wang
et al. 2017)

Glycolysis Metabolism
The phosphorylation level of some glucometabolism enzymes from salt group was
higher than that in the control group, including glycogen phosphorylase (GP),
A pI 3-10 B pI 3-10
293 293
SDS-PAGE
97 95
97 95 100 100
control group salt group
Fig. 11.7 Images of gels stained with SYPRO Ruby for total proteins (a) control; (b) meat salted
with 3% NaCl (Wang et al. 2017)
1.4
control group
Relative protein abundance
1.2
1 salt group
0.8
0.6
0.4
0.2
0
95 97 100 293
The number of protein spots
Fig. 11.8 Quantification of proteins stained by SYPRO Ruby in selected spots which were
different between control and salted meat in protein abundance (the ratio <2/3 or >1.5) (Wang
et al. 2017)
triosephosphate isomerase (TPI), and creatine kinase M-type (CK). The addition of
sodium chloride to these enzymes might change glycolysis in muscle and influence
the meat color, tenderness, and water holding capacity. GP could be divided into
liver type, muscle type, and brain type. GP was one of the glycolytic enzymes which
limited the glycolysis rate. Agius et al. found out that phosphorylase kinase could
phosphorylate GP on serine 14, which transformed the inactive form (GPb) to the
Table 11.2 Proteins identified to be differentially phosphorylated in control and salted muscles
(Wang et al. 2017)
Spot noa Accession nob Protein namesb PIc MPd Seq. Cove
1 P02190 Myoglobin 6.9 15 74%
2 A0JNJ5 Myosin light chain 1/3 4.96 11 41%
10 Q5KR48 Tropomyosin beta chain 4.66 43 89%
18 P68138 Actin(alpha skeletal muscle) 5.23 27 71%
56 Q8MKH8 Troponin T(fast skeletal muscle type) 7.74 25 55%
124 V6F9A2 Apolipoprotein A-I preproprotein 5.71 12 36%
125 Q5E956 Triosephosphate isomerase 6.45 21 87%
142 Q5E997 F-actin-capping protein subunit alpha-2 5.57 8 24%
a
Spot number of the identified proteins
b
c
The isoelectric points of identified proteins
d
e
Table 11.3 Proteins identified to be different in total protein abundance between control and salted
meat (Wang et al. 2017)
Spot noa Accession nob Protein namesb PIc MPd Seq. Cove
95 Q9XSC6 Creatine kinase (M-type) 6.63 30 58%
100 Q9XSC6 Creatine kinase M-type 6.63 30 75%
293 O18751 Glycogen phosphorylase 6.65 25 79%
a
Spot number of the identified proteins
b
c
The isoelectric points of identified proteins
d
e
active form (GPa) (Agius 2015). The protein phosphorylation level of GP of salt
group was higher than the control group, so the enzyme activity of salt group was
higher than the control group. Then glycolysis process was accelerated and produced
acidic components, indirectly influencing the meat color and tenderness (Cottrell
et al. 2008; Huang et al. 2014).
TPI exists in multiple tissues, which is an important isomerase involved in
glycolysis. TPI catalyzes the reversible interconversion of dihydroxyacetone phos-
phate (DHAP) and glucose aldehyde-3-phosphate (G3P) (Hipkiss 2011). Only G3P
can proceed further down the glycolytic pathway (Fonvielle et al. 2005). Phosphor-
ylated TPI was a direct substrate of cyclin-dependent protein kinase 2 (Cdk2). Lee
et al. (2010) discovered that phosphorylation of TPI decreased its enzyme activity,
which was not conducive to the conversion of G3P to DHAP, and then decreased the
rate of glycolysis. In the study, salt decreased the phosphorylation level of TPI,
which was likely to increase the enzyme activity, probably resulting in accelerating
glycolysis process and indirectly improving meat tenderness (Cottrell et al. 2008).
CK ubiquitously exists in skeletal muscles, brain tissues, and other tissues. It is
well known that CK is one of the most critical enzymes regulating ATP generation
and energy metabolism (Quest et al. 1990; Dieni and Storey 2009). Brain-type
creatine kinase can be phosphorylated by AMP-activated protein kinase (AMPK)
on serine 6 (Ramírez Ríos et al. 2014). Muscle-type creatine kinase can be phos-
phorylated by protein kinase A, protein kinase G (PKG), protein kinase C (PKC),
and AMPK. Phosphorylation of CK increased its activity, whereas dephosphoryla-
tion decreased its activity (Dieni and Storey 2009). The relative protein abundance of
CK stained by SYPRO Ruby in salt group was much higher than without salt
addition, indicating the phosphorylation of CK might decrease and retard enzyme
activity. The reduction of CK activity probably lowered the ATP generation from
phosphocreatine and ADP, with the result that ATP was only produced from
glycolysis metabolism (Ishida et al. 1994; Grehl et al. 1998).
11.3.3 Differential Phosphorylated Proteins Changing

the Protein Function
Myoglobin was also the differential phosphoprotein between the two groups, whose
phosphorylation level was higher in the salt group. On the other hand, it was well
known that sodium chloride caused the muscle or meat paste discoloration, hence,
the phosphorylation level of myoglobin was negative correlated with meat color
(Kanner et al. 1991). Huang also found out that myoglobin could be modified by
phosphorylation (Huang et al. 2011). Canto et al. further discovered that there were
4 differential phosphorylated myoglobin isoforms by 2-DE gels (Canto et al. 2015),
which was due to phosphorylation modification would change myoglobin pI (Zhu
et al. 2005). Thus, the phosphorylation modification of myoglobin might actually
influence meat color stability and was more likely to make the meat dark or brown,
which might be due to phosphorylation accelerated the iron release from myoglobin
and tended to be oxidized (Devatkal and Naveena 2010).
Apolipoprotein A-I preproprotein (APOA1) is part of high-density lipoprotein
(HDL), which carries cholesterol from tissues to liver, and associated with protection
against cardiovascular disease. APOA1 reduces cardiovascular disease significantly,
even though it causes a reduction in HDL level and an increase in triglyceride level
(Chistiakov et al. 2016; Midtgaard et al. 2015). APOA1 exists in muscle but there is
no well understanding of its function related to phosphorylation modification. The
salting treatment decreased APOA1’s phosphorylation in muscle, but its function to
meat quality (lipid oxidation, lipolysis) and meat nutrition should be further
investigated.

the Muscle Contractile and Protein Dissociation
The differential phosphorylated proteins identified between salt group and control
group included TNT (56, 70, 97), actin (18, 62, 84), TPM2 (10), CAPZA2 (142), and
MLC1 (2, 20, 81), while these proteins were all belonged to myofibrillar proteins.
Actin, TPM2, and MLC1 might affect the muscle contractile, and TNT, TPM2, and
CAPZA2 might influence actomyosin dissociation. The phosphorylation of these
myofibrillar proteins reduced their degradation by calpains, and maintained the
sarcomere texture (Zhang et al. 1988; Di Lisa et al. 1995). According to the relative
phosphorylation (gray level of 56 and 70 in Fig. 11.5, divided by gray level of 97 in
Fig. 11.7), it indicated that salt curing increased the phosphorylation level of TNT
significantly. TNT contained several phosphorylation sites, while serine 2 was
highly phosphorylated (Katrukha and Gusev 2013). The phosphorylation of TNT
induced by protein kinase C made it more easier to be degraded by calpains (Toyo-
oka 1982). The relative phosphorylation level of TNT in salt group was much higher
than in no salt group, resulting in TNT being degraded easily by calpain.
Tropomyosin (TPM2) is an important protein for regulating muscle contraction
and the most important phosphorylated protein in the muscle filament. Lehman
found out that tropomyosin was phosphorylated at serine 283 position, then
enhanced the binding of the head and tail of tropomyosin (Lehman et al. 2015).
The phosphorylation of TPM2 was necessary for long-range cooperative activation
of myosin binding (Rao et al. 2009). The phosphorylation of TPM2 in 3% salt group
was higher than 0% control group, which might strengthen the binding with actin or
myosin, resulting in slowing the actomyosin dissociation.
Myosin and actin were the main components of myofibrillar proteins, while
myosin regulatory light chain could change myosin conformation and regulate the
binding of myosin with actin. Myosin light chain 1/3 was myosin regulatory light
chain. Some authors reported that MLC1’s phosphorylation would inhibit actomy-
osin dissociation, which quickened muscle contraction and restrained the myofibril
structure (Miller et al. 2011; Alamo et al. 2008). Other authors reported that actin
could be phosphorylated and dephosphorylated on tyrosine residues (Gauthier et al.
1997). Phosphorylation of actin made it difficult to be degraded by μ-calpain
(Li et al. 2017), resulting in good texture of meat during the salting. Similar to
actin, F-actin-capping protein subunit alpha-2 (CAPZA2) was also a kind of myo-
fibrillar proteins, which bound in a calcium-independent manner to the fast growing
ends of actin filaments (barbed end), thereby restricting its growth or dissociation
(Hart et al. 1997). The phosphorylation level of CAPZA2 was lower in salt group,
which might reveal that salt weakened the binding of CAPZA2 and actin through
phosphorylation modification.
Salt curing influences phosphorylation and dephosphorylation of muscle pro-
teins, which accelerates glycolysis metabolism, weakens the protein positive func-
tion to meat quality, and regulates the muscle contractile to cause the actomyosin
dissociation. Probably, phosphorylation modification altered by salt curing might be
11.4 Effect of Salting Temperature on the Protein Phosphorylation of Muscle 257
a new mechanism to regulate meat quality, especially the meat color and tenderness.
In the future, the regulation mechanism of muscle protein function by phosphoryla-
tion under sodium chloride should be expounded, moreover, some essential methods
were supposed to be invented.
11.4 Effect of Salting Temperature on the Protein

Phosphorylation of Muscle
The effect of different salting temperature on the phosphorylation level of myofi-

brillar and sarcoplasmic proteins in mutton was investigated. The topside muscles
from both hind legs were removed immediately after slaughter and aged at 4 C for
24 h, and then three salting treatment groups were set up at 1 C, 4 C, and 25 C.
Samples from each group were collected at 0, 8, 16, and 24 h, and sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with fluores-
cence staining were employed to analyze phosphorylation level. The results showed
that the effect of salting temperature on the phosphorylation level of individual
protein bands was different, while salting temperature could regulate the meat
quality by influencing the phosphorylation of muscle proteins.
11.4.1 Effect of Salting Temperature on the Myofibrillar

The SDS-PAGE electrophoresis of myofibril protein after curing for 0, 8, 16, and
24 h at freezing temperature (1 C), 4 C and room temperature (25 C) was shown
in Fig. 11.9. Figure 11.9a shows the phosphorylated myofibril protein with Pro-Q
Diamond staining, and Fig. 11.9b shows the whole myofibril protein with SYPRO
Ruby staining. As shown in Fig. 11.9, the separation effect of protein bands was
better. The relative optical density of each band was analyzed by the Quantity One
4.6.2 software to obtain the P/T ratio, so as to obtain the changes in the total
phosphorylation level of myofibril protein during the curing process at different
temperature.
Changes in the total phosphorylation level of myofibril protein during the curing
process at different temperatures were shown in Fig. 11.10. As can be seen from the
figure, the total phosphorylation level of myofibril protein in the ice temperature
treatment group was higher than that in the 4 C group, while that in the 4 C group
was higher than that in the room temperature treatment group. However, the ice
temperature treatment group was significantly higher than the room temperature
treatment group at 8 h of curing (P < 0.05), and there was no significant difference
among the three treatment groups at 16 and 24 h of curing. The results indicated that
low curing temperature was conducive to maintaining the total phosphorylation level
8h 16 h 24 h 8h 16 h 24 h
M 0h -1 4 25 -1 4 25 -1 4 25（ºC） M 0h -1 4 25 -1 4 25 -1 4 25（ºC）
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11 11
12 12
13 13
14 14
15 15
16 16
17
17
18
18
19
19
20 20
21 21
A B
Fig. 11.9 Gel images of phosphoproteins and total myofibrillar proteins of different salting
temperature separated by SDS-PAGE. (a) Image of phosphorylated myofibrillar proteins by
Pro-Q Diamond staining; (b) Image of total myofibrillar proteins by SYPRO Ruby staining
(Zhang et al. 2016b)
0.6
0.5
a
0.4
ab b
P/T value
0.3
0.2
0.1
0
0h 8h 16 h 24 h
salting time˄h˅
Fig. 11.10 Phosphorylation of myofibrillar proteins during different salting temperature. Different
letters at the top of the bar indicate significant difference (P < 0.05). Adapted from (Zhang et al.
2016b)
of myofibril protein. After curing, the total phosphorylation level of myofibril

protein was significantly lower than that of raw meat at 0 h, and the protein
phosphorylation level of all three groups showed a downward trend after curing,
indicating that salt reduced the total phosphorylation level of myofibril protein,
which was consistent with the experimental results in Chap. 2.
Four important myofibrillar protein bands (band 3, 4, 11, and 21) were selected to
analyze the effect of curing temperature on its phosphorylation level, as shown in
Fig. 11.11.The phosphorylation level of band 3 in the ice temperature treatment
group was higher than that in the 4 C treatment group at 8, 16, and 24 h after curing,
and that in the 4 C treatment group was higher than that in the room temperature
treatment group, but there was no significant difference between the different
treatment groups. However, the phosphorylation level of band 21 at 8, 16, and
24 h after curing was significantly higher in the ice temperature treatment group
than in the 4 C treatment group, as well as in the 4 C treatment group than in the
room temperature treatment group. This indicated that the curing temperature was
within the range of ice temperature to room temperature, and the lower the curing
temperature was, the better the phosphorylation level of band 3 and 21 would
be. The phosphorylation level of band 4 and 11 was opposite to that of band 3 and
21. The phosphorylation level of band 11 in the room temperature treatment group
was higher than that in the ice temperature treatment group, but there was no
significant difference. The phosphorylation level of band 4 in the room temperature
group was significantly higher than that in the ice temperature group and 4 C
treatment group (P < 0.05). It was indicated that the higher the curing temperature,
the more beneficial the improvement of the phosphorylation level of band 4 and 11.
Overall, the whole phosphorylation level of myofibrillar protein in the ice tem-
perature group was significantly higher than that in the other two groups, and the
effect of curing temperature on the phosphorylation level of different protein bands
was different. According to the mass spectrometry identification results of Chen and
Huang et al. (Chen et al. 2016; Huang et al. 2012), the phosphorylation level of some
important myofibrillar protein bands was selected and analyzed, as shown in
Fig. 11.11. Band 4 was mainly myosin binding protein C (MYBPC), and MYBPC
phosphorylation affected muscle contraction through specific sites. Wang et al.
(2014) found out that MYBPC was an allosteric activator of myosin, thus enhancing
the formation of transverse bridging force. The phosphorylation of MYBPC could
regulate this transverse bridging force and accelerate the formation of contraction
force (Wang et al. 2014). Band 21 was mainly myosin light chain. The phosphor-
ylation of myosin light chain was regulated by many enzymes, such as myosin
regulated light chain kinase (MLCK), protein kinase A, and protein kinase C (Ikebe
et al. 1999). Many studies have proved that the phosphorylation of myosin light
chain was related to muscle contraction (Farman et al. 2009; Taylor et al. 2014).
Phosphorylation can change the structure of myosin light chain, and phosphorylation
of myosin light chain 2 can increase the number of strong binding transverse bridges
of actin (Miller et al. 2011). The degree of contractility enhancement was positively
correlated with the degree of myosin light chain 2 phosphorylation (Stull et al.
2011). In this experiment, the phosphorylation level of myosin light chain in the
ice temperature group was significantly higher than that in the other two groups, and
the muscle contraction was the strongest, which was not conducive to the formation
Fig. 11.11 The band 3 (myosin heavy chain)

phosphorylation level of
1.0
important myofibrillar
proteins in different 0.9
temperature groups.
0.8
P/T value
Different letters at the top of
the bar indicate significant 0.7
difference (P<0.05).
Adapted from (Zhang et al. 0.6
2016b)
0.5
0h 8h 16 h 24 h
salting time (h)
band 4 (Myosin binding protein C)

1.2
a
1.0
0.8 a
P/T value
a
b b b b
0.6 b b
0.4
0.2
0.0
0h 8h 16 h 24 h
salting time (h)
band 11 (actin)
0.30
0.25
P/T value
0.20
0.15
0.10
0.05
0.00
0h 8h 16 h 24 h
salting time (h)
band 21 (myosin light chain)

2.5
2.0
P/T value
a
1.5 a
a
b b
1.0 b b
c c
0.5
0.0
0h 8h 16 h 24 h
salting time (h)
of tenderness. In addition to the above proteins, band 3 was mainly myosin heavy
chain and band 11 was mainly actin, and the phosphorylation level of the above two
bands was not significantly different among the three treatment groups (P > 0.05).
Studies have found that protein phosphorylation was not conducive to protein
degradation by calpain (Zhang et al. 1988, 2012). Calpain in muscle can promote
meat tenderization by degrading myofibrillar protein. However, the whole phos-
phorylation level of myofibrillar protein in the ice temperature salting group was
high, which was not conducive to the degradation of myofibrillar protein and the
tenderization of meat. The whole phosphorylation level of myofibrillar protein was
the lowest at room temperature. Compared with the other two treatment groups, the
protein was degraded to a large extent, which was conducive to the tenderization of
meat. Therefore, it was of great significance to study the effect of different temper-
ature on protein phosphorylation in muscle to regulate meat quality.
11.4.2 Effect of Salting Temperature on the Sarcoplasmic

The SDS-PAGE electrophoresis patterns of sarcoplasmic proteins salted at ice

temperature (1 C), 4 C and room temperature (25 C) for 0, 8, 16, and 24 h
were shown in Fig. 11.12. Figure 11.12a shows phosphorylated sarcoplasmic pro-
teins stained with Pro-Q Diamond, and Fig. 11.12b shows total sarcoplasmic pro-
teins stained with SYPRO Ruby. Twenty clear protein bands were selected from the
gel for total phosphorylation level analysis, so did the myofibril protein.
The total phosphorylation level of sarcoplasmic proteins during salting at differ-
ent temperature was shown in Fig. 11.13. The total phosphorylation level of sarco-
plasmic protein in the three temperature treatment groups was different only at 8 h,
but there was no significant difference at 16 and 24 h. After salting for 8 h, the total
phosphorylation level of sarcoplasmic protein in ice temperature group was signif-
icantly lower than that of 4 C group and room temperature group (P < 0.05), but
there was no significant difference between 4 C group and room temperature group
(P < 0.05).
According to the results of electropherogram, four protein bands (band 14, 15,
16, and 19) with dark color and obvious gray value were selected to analyze the
effect of different salting temperature on their phosphorylation level. The results
were shown in Fig. 11.14. The total phosphorylation level of the four bands was
affected by salting temperature. The P/T values of band 14 and 19 showed the same
overall trend. The ice temperature group was significantly higher than the 4 C group
(P < 0.05), and the 4 C treatment group was significantly higher than the room
temperature group (P < 0.05). The results showed that when the salting temperature
was in the range of ice temperature to room temperature, the lower the salting
temperature was, the more favorable it was to maintain the phosphorylation level
of band 14 and 19. The P/T value of band 15 in the room temperature group was
-1 ºC 4 ºC 25 ºC -1 ºC 4 ºC 25 ºC
M 0h 8h 16h 24h 8h 16h 24h 8h 16h 24h M 0h 8h 16h 24h 8h 16h 24h 8h 16h 24h
1 1
2 2
3 3
4 4
5 5
6 6
7 7
8 8
9 9
10 10
11 11
12 12
13
14 13
14
15 15
16 16
17 17
18 18
19
19
20 20
A B
Fig. 11.12 Gel images of phosphoproteins and total sarcoplasmic proteins of different salting
temperature separated by SDS-PAGE. (a) Image of phosphorylated sarcoplasmic proteins by Pro-Q
Diamond staining; (b) Image of total sarcoplasmic proteins by SYPRO Ruby staining (Zhang et al.
2016b)
0.5
a a
-1đ
b
0.4 4đ
25đ
0.3
P/T value
0.2
0.1
0
0h 8h 16 h 24 h
salting time（h）
Fig. 11.13 Phosphorylation of sarcoplasmic proteins during different salting temperature. Differ-
ent letters at the top of the bar indicate significant difference (P<0.05). Adapted from (Zhang et al.
2016b)
Fig. 11.14 The band 14 (enolase)

phosphorylation level of 0.6
sarcoplasmic myofibrillar 0.5 a a
a a
proteins in different b
0.4
P/T value
temperature groups.
b
Adapted from (Zhang et al. 0.3
2016b)
0.2
c
0.1 c
c
0
0h 8h 16h 24h
salting time (h)
band 15 (aldolase)
1.8 a
a
a b
1.5 b b
P/T value
c c
1.2
c
0.9
0.6
0.3
0
0h 8h 16h 24h
salting time (h)
band 16 (lyceraldehyde-3-phosphate
dehydrogenase)
0.4
a
a
a
ab b
0.3 b
b
b
P/T value
0.2
0.1
0
0h 8h 16h 24h
salting time (h)
band 19 (adenylate kinase)

1
a a
0.8 a
a
P/T value
0.6
b b
b b
c
0.4
0.2
0
0h 8h 16h 24h
salting time (h)

significantly higher than the 4 C group (P < 0.05), and in the 4 C group was
significantly higher than the ice temperature group (P < 0.05), which was opposite
to that of band 14 and 19. When being salted for 16 h or 24 h, the band 16 in the 4 C
group was significantly higher than that in the other two groups (P < 0.05).
The results showed that the total phosphorylation level of sarcoplasmic protein in
ice temperature treatment group was lower, but the effect of salting temperature on
the phosphorylation of different sarcoplasmic protein bands was significantly dif-
ferent. According to the mass spectrometric results of sarcoplasmic proteins in the
second chapter, the protein names of the four selected protein bands were deter-
mined. Band 14 may be enolase. Enolase was a rate-limiting enzyme in the pathway
of postmortem muscle glycolysis, which catalyzed the transformation between
2-phosphoglyceric acid and phosphoenolpyruvate. The level of phosphorylation
decreased significantly with the increase of salting temperature (P < 0.05). The
results showed that high temperature promoted the dephosphorylation of enolase.
Band 15 may be aldolase, and its phosphorylation level increased with the increase
of salting temperature. Aldolase was also an important enzyme in the process of
glycolysis, and the change of its phosphorylation level may in turn regulate the
process of glycolysis by affecting the activity of the enzyme. Band 16 may be
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was an intermediate
enzyme in glycolysis. The activity of GAPDH decreased after phosphorylation,
which was not conducive to glycolysis (Bell and Storey 2014). As can be seen
from the electropherogram Fig. 11.12b, the gray value of band 14 decreased with the
increase of salting time and salting temperature, indicating that the degradation
degree of band 14 increased. The band 19 showed the opposite phenomenon, and
its gray value increased with the increase of salting time and salting temperature, so it
was speculated that band 19 was probably the degradation product of band 14.
The optimum temperature of intestinal alkaline phosphatase (AP) of silver carp
was 25 C (Xu 2007). Therefore, the activity of AP was the highest at room
temperature, and dephosphorylation was more likely to occur. Band 14 had the
lowest level of phosphorylation at room temperature, indicating that alkaline phos-
phatase played a full role in the dephosphorylation of enolase, resulting in a low
level of phosphorylation. The highest phosphorylation level of band 15 at room
temperature indicated that aldolase might not be the substrate of alkaline phospha-
tase. Other studies have also discovered that temperature also affected the activity of
protein kinases. For example, the activity of AMP-activated protein kinase (AMPK)
increased with the increase of temperature at 20–32 C, but decreased at 32–36 C
(Jost et al. 2015). Therefore, the effect of salting temperature on protein phosphor-
ylation was mainly due to the effect of salting temperature on the activities of protein
kinase and phosphatase.
Enzyme phosphorylation, especially glycolytic enzyme phosphorylation, can
change the activity and stability of the enzyme itself, but the effects of phosphory-
lation on the activity of different enzymes are different (Huang et al. 2011). The
results showed that the lower the salting temperature was, the lower the total
phosphorylation level of sarcoplasmic protein was, but the effect on the phosphor-
ylation level of different enzymes was different. It was speculated that the
11.5 Conclusions 265
phosphorylation of proteins was regulated by different kinases. The tolerance of

these kinases to temperature was not the same. Combined with the results of
previous studies, temperature played an important role in meat quality by affecting
protein phosphorylation and regulating enzyme activity, and then affecting glycol-
ysis and protein degradation.
In summary, salting temperature will affect the phosphorylation level of myofi-
brillar protein and sarcoplasmic protein. Protein phosphorylation is regulated by a
variety of protein kinases and phosphatases, and there are many kinds of protein
kinases (about 300), and phosphatase also has a variety of isozymes. Temperature is
an important factor in enzyme activity. Different enzymes have different optimum
temperatures (Daniel and Danson 2013), and protein phosphorylation is regulated by
many kinds of protein kinases and phosphatases; therefore, the phosphorylation
level of different protein bands is affected by temperature. Salting temperature
affects the level of protein phosphorylation by altering the activity of enzymes
related to protein phosphorylation, thus further regulating the quality of salted meat.
The phosphorylation level of muscle protein was affected by curing temperature.
The whole phosphorylation level of myofibrillar protein was the highest under ice
temperature. The lower the curing temperature, the higher the phosphorylation level.
The global phosphorylation level of myofibrillar protein was the highest under room
temperature. The phosphorylation level decreased with the descent of curing tem-
perature. The effect of temperature on the phosphorylation level of individual protein
bands was different. The mechanism of protein phosphorylation at different curing
temperatures on meat quality needs to be further investigated, and on this basis, a
new curing technology based on muscle protein post-modification should be studied.
11.5 Conclusions
Salt concentration and environmental temperature influenced phosphorylation and

dephosphorylation of muscle proteins during salting. Higher ionic strength of salt
and higher salting temperature decreased the global phosphorylation level of myo-
fibrillar proteins and influenced the phosphorylation level of individual sarcoplasmic
protein, which accelerated glycolysis metabolism, weaken the protein positive
function of meat quality, and regulated the muscle contractile to cause the actomy-
osin dissociation. Therefore, salt curing altering protein phosphorylation might be a
new mechanism to regulate meat quality, especially the color and tenderness of meat.
The study may provide a new insight into the mechanism by which salting influences
the quality of processed meat products, especially the meat tenderness which is
related to protein degradation and muscle contraction.
Acknowledgments Parts of this chapter are reprinted from Meat Science, 133, Wang Z. et al.,
Comparative analysis of muscle phospho-proteomics induced by salt curing, 19–25; Food Science
and Biotechnology, 25, Zhang C. et al., Phospho-proteomic profiling of myofibrillar and sarco-
plasmic proteins of muscle in response to salting, 993–1001, Copyright (2020), with permission
from Elsevier. Parts of this chapter are translated from Modern Food Science and Technology,
32, Zhang C. et al., Effect of Salting Temperature on the Protein Phosphorylation of Mutton
(Chinese), 215–221. Copyright (2020), with permission from Journal of Modern Food Science
and Technology.
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Chapter 12
Improvement of Meat Quality by Novel
Technology
Abstract Developing new techniques to improve meat quality is a popular topic. In

this chapter, three novel methods for improving the meat quality are provided,
including controlled freezing point storage (CFPS), low-variable temperature and
high humidity thawing (LVTHHT), and pulsed vacuum pickling (PVP) technolo-
gies. Meat stored in CFPS treatment for 10 days proved to have better color stability
in comparison with those in 4 C storage. The LVTHHT could significantly prevent
the deterioration of lamb quality, and it was low cost and easy to operate. PVP could
improve salting efficiency and meat quality.
Keywords Controlled freezing point storage · Low-variable temperature and high

humidity thawing · Pulsed vacuum pickling · Meat quality · Novel technology
12.1 Introduction
Controlled freezing point storage (CFPS), also known as subzero or super chilling
storage, is an alternative and promising technique for preservation of fruit, vegetable,
and meat products without freezing. Compared to freezing, it provides a storage
condition that can lead to a significant retardation of biochemical and microbial
activity of muscle tissue without physically damaging the muscle cells due to ice
crystal formation. In contrast with regular 4 C refrigerated storage, CFPS has the
characteristics of fast chilling rate, stemming from the high heat exchange capacity,
as well as small temperature variation range because of strict control. Due to
production factors such as low product turnover rate, meat products often go through
to a long production chain. The CFPS condition offers great flexibility for meat
retailers and processors to meet the requirement of this situation in comparison with
refrigerated storage and freezing.
Freezing storage is the most important storage method for meat. Frozen meat is
also the main form of meat products import and export trade and inter-regional
circulation (Huang 2005). At present, the thawing methods of frozen meat mainly
include air thawing, water immersion thawing, microwave thawing, ultra-high
pressure thawing, and the like. The existing methods have their own advantages

https://doi.org/10.1007/978-981-15-9441-0_12
272 12 Improvement of Meat Quality by Novel Technology
and disadvantages. The conventional air thawing is currently the most widely used
method, which has the advantages of simple operation and low cost and the
disadvantages of slow thawing speed, easy oxidation and discoloration on the
meat surface, and rapid proliferation of microorganisms. Thawing speed of water
immersion thawing is faster than the air thawing, but easy to cause loss of nutrients
and make meat gray (Yang 2005). Microwave thawing possesses high speed and
efficiency, but due to the difference of microwave penetration and absorption of
water and ice, it is easy to generate uneven thawing of meat and local overheating
(Xia et al. 2012). Ultra-high pressure thawing method can thaw quickly and has little
negative impact on product quality, whereas both the requirement and cost of the
equipment are higher than the other methods, so it is still immature in domestic
industrialization (Liao et al. 2010). Therefore, it is of great significance for the meat
industry to develop new and economical thawing techniques that can maintain the
quality of thawed meat and solve the problems of juice loss, oxidation, and texture
deterioration during the process.
The pickling processing is an important technology for processing meat products,
but the traditional pickling (dry curing, wet curing, mixed curing) has many defects,
such as tedious processes, long processing time, and hygiene and quality risk.
Modern pickling techniques include brine injecting, vacuum pickling, pulsating
pickling, and tumbling pickling. Pulse pressure salting technology can be applied
to the processing of meat products completely immersed in the marinade. The main
reason for the pulse pressure salting to improve the pickling effect is that the
hydrodynamic mechanism (Fito, 1994) promotes the pickling liquid from the
pores of the meat during the pressure change (Guamis et al. 1997), and the cyclical
change of pressure can improve the pickling effect. Pulse pressure salting is an
efficient static pickling method, which can effectively improve the pickling rate and
better maintain the meat form (Deumier et al. 2011). In recent years, researches on
pulse pressure salting have focused on the diffusion of salt and water, for example,
previous studied the law of water change of sardine during pulse pressure salting
(Corzo and Bracho 2007; Corzo et al. 2006b; Reyes et al. 2008), and Deumier et al.
(2011) studied the mass transfer mechanism of turkey meat during pulse pressure
salting. Nevertheless, there has been little research on meat quality using pulse
pressure salting technology and no report on mutton pickling.
In this chapter, the CFPS, low-variable temperature and high humidity thawing
(LVTHHT), and pulsed vacuum pickling (PVP) technologies were introduced,
which can provide technical guidance to produce high quality meat.
12.2 Controlled Freezing Point Storage
This study investigated the mechanism of controlled freezing point storage on

muscle color and protein phosphorylation level. The 20 longissimus dorsi of
10 crossbred sheep were used. The two longissimus dorsi of the same sheep were
randomly divided into two groups, and each longissimus dorsi was equally divided
12.2 Controlled Freezing Point Storage 273
Fig. 12.1 The freezing point of ovine muscle. Translated from Zhang (2016)
and then randomly assigned to each time point. Compared with refrigeration, the
effects of controlled freezing point on muscle color and protein phosphorylation
level during storage time were studied. In order to estimate the color stability of meat
stored at different temperatures, a Konica Minolta colorimeters CM-600d was used
to measure instrumental color and reflectance of samples at different wavelengths.
The activity of metmyoglobin reductase and concentration of nicotinamide adenine
dinucleotide (NADH) were determined by spectrophotometry, then analyzed the
mechanism of the effect of controlled freezing point storage on color stability. The
activity of protein kinase was tested by enzyme linked immunosorbent assay
(ELISA). In order to investigate the phosphorylation level of sarcoplasmic protein
and myofibrillar protein in postmortem ovine muscle at different storage tempera-
tures, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
coupled with Pro-Q Diamond and SYPRO Ruby staining was applied. Quantity
One software was used to analyze the optical density of protein bands on
SDS-PAGE images. By analyzing the changes of color and protein phosphorylation
during the CFPS muscle and cold storage conditions, the effects of CFPS on color
stability and protein phosphorylation level were investigated, which provided a
theoretical basis to study the mechanism of storage temperature controlling meat
quality. The research also offered a reference frame to companies and consumers to
produce and select ovine muscle with high quality.
As shown in Fig. 12.1, the temperature measurement time of the sample was
85 min, and the maximum ice crystal formation area was around 15–40 min. The
temperature fluctuation was small, and the sample stayed in this temperature zone for
a long time. According to the temperature recorder test data, the sample temperature
continued to decrease before 22 min, a slight rebound occurred at 22 min, from 2 to
1.9 C, then decreased to 2 C at 25 min, then again at 27 min. The temperature
raised to 1.9 C and then continued to decrease, so the freezing point of the
experimental sample was determined to be 2 C. Considering the temperature
fluctuation range of the incubator with an accuracy of 1 C, to prevent the sample
from freezing, the temperature of CFPS was set to 0.8 C.
12.2.1 Effect of Controlled Freezing Point Storage on Meat

Color Stability
Figure 12.2 showed the color difference values of samples stored at day 0, 2, 4, 6,
8, and 10, respectively, under CFPS and refrigerated conditions, in which
Fig. 12.2a–c were graphs showing changes of brightness value L*, redness value
a*, and yellowness value b* with storage time. As shown in Fig. 12.2a, the L* value
of the two treatment groups had the same change in the storage process, which was a
trend of first rising, then decreasing and then stabilizing, and the highest brightness
values appeared in the day 2–4. During the whole storage process, the L* value of the
group of CFPS fluctuated greatly, with day 2, 4, 6, 8, and 10 significantly higher than
day 0 (P < 0.05), and day 2 and 4 significantly higher than day 6 and 10. There was
no significant difference between day 2 and day 4, 6, and 10. The L* value of the cold
storage group did not change much, and that of day 2, 4, 6, 8, and 10 was
significantly higher than day 0 (P < 0.05), while there was no significant difference
between day 2, 4, 6, 8, and 10. At day 2, 4, and 8, the L* value of the group of CFPS
was significantly higher than that of the refrigerated group (P < 0.05).
Fig. 12.2 Changes of instrumental meat color at different storage temperature. The lowercases
represent the significant difference between different postmortem time of refrigeration (P < 0.05),
while the capital letters represent the significant difference between different postmortem time of
controlled freezing point (P < 0.05). * represents the significant difference between controlled
freezing point and refrigeration at the same time of storage (P < 0.05). (a) Changes of brightness
value L* with storage time, (b) changes of redness value a* with storage time, (c) changes of
yellowness value b* with storage time. Translated from Zhang (2016)
As shown in Fig. 12.2b, at the initial stage of storage (0–2 days), the a* values of
the two treatment groups showed an upward trend, and then the a* value of the CFPS
group was stable, while the a* value of the refrigerated group gradually decreased
from the day 6 of storage. Both the maximum a* values of CFPS and refrigerated
groups appeared at day 0. Compared with the refrigerated group, the a* value of the
CFPS group fluctuated greatly during the whole storage process, and only that of day
2, 4, 6, 8, and 10 was significantly higher than day 0, while there was no significant
difference at day 2, 4, 6, 8, and 10 (P < 0.05). The a* value of the cold storage group
was significantly higher than day 0, 6, 8, and 10 at day 2 and day 4 during storage.
From the day 2 of storage to the end of the storage period, the a* value of the CFPS
group was always significantly higher than that of the refrigerated group (P < 0.05).
As shown in Fig. 12.2c, during the storage process, the b* values of the CFPS and
refrigerated treatment samples showed the same changing pattern as the a* values,
that is, first increased and then decreased, the maximum value of CFPS group
appeared on the day 2, decreased on the fourth day, then stabilized. The maximum
value of the refrigerated storage group appeared on the day 2 and 4, decreased on the
sixth day, then showed a steady state. During the whole storage process, the b* value
of the CFPS group was significantly higher than day 0 at day 2, 4, 6, 8, and 10, day
2 was significantly higher than day 4, 6, 8, and 10, and there was no significant
difference on day 4, 6, 8, and 10 (P < 0.05). The b* value of the control group was
also significantly higher than day 0 at day 2, 4, 6, 8, and 10, day 2 and 4 was
significantly higher than day 6, 8, and 10, and there was no significant difference
between day 6, 8, and 10 (P < 0.05). From day 2 till the end of the storage period, the
b* value of the CFPS group was always significantly higher than that control group
(P < 0.05).
As shown in Fig. 12.3, compared with the refrigerated group, the R630/580 value
in the CFPS group was relatively stable, with the trend of first increasing and then
decreasing. R630/580 value of the control group was first lowered and then
increased. The R630/580 value of CFPS group was significantly higher on day
2, 4, and 6 than day 0, 8, and 10, and there was no significant difference between
day 2, 4, 6 and day 0, 8 (P < 0.05). In the control group, the R630/580 value of day
2 was significantly higher than day 4, 6, 8, 10, day 0 was significantly higher than
day 6, 8, 10. But there was no significant difference of the R630/580 value between
day 0 and day 2, day 0 and day 4, day 6 and day 8, 10, t (P < 0.05). During the whole
storage process, from day 2 to the end of storage, the R630/580 value in the CFPS
group was significantly higher than that in the control group (P < 0.05).
According to Fig. 12.4, during the storage process, the CFPS group was consis-
tent with the changing trend of Chroma value in the control group, both of which
increased first and then slowly decreased. The CFPS group was significantly higher
than 4 C for day 2, 4, 6, 8, and 10, and day 2 was significantly higher than day 8 and
10. There was no significant difference between day 2, 4, and 6, and also had no
significant difference in day 8 and 10 (P < 0.05). The day 2 and 4 of the control
group was significantly higher than day 0, 6, 8, and 10. No difference was observed
between day 2 and 4, and day 6 was significantly higher than day 0. There was no
significant difference between day 6, 8, and 10 (P < 0.05). From day 4 to the end of
Fig. 12.3 Changes of R630/580 value at different storage temperature. The lowercases represent
the significant difference between different postmortem time of refrigeration (P < 0.05), while the
capital letters represent the significant difference between different postmortem time of controlled
freezing point (P < 0.05). * represents the significant difference between the controlled freezing
point and refrigeration at the same time of storage (P < 0.05). Translated from Zhang (2016)
Fig. 12.4 Changes of Chroma value at different storage temperature. The lowercases represent the
significant difference between different postmortem time of refrigeration (P < 0.05), while the
the storage period, the Chroma value of CFPS group was significantly higher than
the control group (P < 0.05).
As shown in Fig. 12.5, Hue angle value of the refrigerated group increased
gradually during the whole storage process. As for the CFPS group, Hue angle
value first increased, then decreased and then stabilized. The maximum value of the
Fig. 12.5 Changes of Hue angle value at different storage temperature. The lowercases represent
the significant difference between different postmortem time of refrigeration (P < 0.05), while the
CFPS group appeared on the day 2 of storage, significantly higher than the others. In
addition, the day 4, 6, 8, and 10 was significantly higher than day 0, and there was no
significant difference between day the 4, 6, 8, and 10. The day 10 of the control
group was significantly higher than the rest of the time points, and day 2, 4, 6, and
8 was significantly higher than day 0, and day 2, 4, 6, 8 had no significant difference.
Hue angle values of the refrigerated group from the day 4 to the day 10 were
significantly higher than those in the CFPS group (P < 0.05).
12.2.2 Effect of Controlled Freezing Point Storage on Protein

Kinases’ Activities
As shown in Fig. 12.6, protein kinase activity increased significantly (P < 0.05) at
0.5–12 h after controlled freezing point storage, and then gradually decreased. The
protein kinase activity in the refrigerated storage process decreased significantly with
the storage time (P < 0.05), and the reduction rate in the refrigerated storage group
was higher than that in the controlled freezing point storage. The protein kinase
activity in the CFPS group was significantly higher than the control group during the
storage period of 12 h–9 days (P < 0.05). At the end of storage, the protein kinase
activity in the control group was close to inactivation and the CFPS group was still at
a higher level.
Protein kinases are a class of enzymes that catalyze the phosphorylation of
proteins, and they play an important role in almost every cell function (Gonz
Lez-Mart Nez et al. 2002). All protein kinases are in the ground state and can be
Fig. 12.6 Analysis of the protein kinases’ activities at different storage temperature. The lower-
cases represent the significant difference between different postmortem time of refrigeration
(P < 0.05), while the capital letters represent the significant difference between different postmor-
tem time of controlled freezing point (P < 0.05). * represents the significant difference between the
controlled freezing point and refrigeration at the same time of storage (P < 0.05). Translated from
Zhang et al. (2016)
activated by different stimuli when needed, catalyzing the phosphorylation of certain

proteins and activating phosphatases (Roskoski 2015). Kemp et al. (2006) have
shown that AMP-activated protein kinase (AMPK) in pale, soft, and exudative (PSE)
meat is activated in the early postmortem period and has the highest enzyme activity
at 0.5 h, then gradually decreases, while AMPK enzyme activity in normal pork has
the highest enzyme activity at 1 h, indicating that AMPK activity has an effect on the
quality of postmortem meat. AMPK plays a key role in the postmortem cell
glycolysis process and the formation of PSE meat. The consumption of adenosine
triphosphate (ATP) and the production of adenosine monophosphate (AMP) in the
early postmortem period cause AMPK to be activated, resulting in a rapid decline in
pH of muscle, and in pork, it is the formation of PSE meat (Baron et al. 2005; Hwang
et al. 2004). Li et al. (2016) found that the muscles stored at controlled freezing point
reached their ultimate pH later 4 days than the control. In this study, the activation of
protein kinase activity under controlled freezing point conditions was lagged behind
the refrigeration, and together with the study of Li, we are able to reveal the reasons
for the longer time required for muscles to reach the ultimate pH under CFPS
conditions.
12.2.3 Effect of Controlled Freezing Point Storage

on Phosphorylation Levels of Sarcoplasmic Protein
The SDS-PAGE electrophoresis patterns of sarcoplasmic proteins in the CFPS group

and refrigerated group at 0.5 h, 12 h, 24 h, 3 d, 5 d, and 9 d after postmortem were
Fig. 12.7 Gel images of phosphoproteins and total proteins of sarcoplasmic protein at different
storage temperature separated by SDS-PAGE. (a) Image of phosphoproteins stained with Pro-Q
Diamond. (b) Image of total proteins stained with SYPRO Ruby. Translated from Zhang et al.
(2016)
Fig. 12.8 The global phosphorylation level of sarcoplasmic protein at different storage tempera-
ture. The lowercases represent the significant difference between different postmortem time of
refrigeration (P < 0.05), while the capital letters represent the significant difference between
different postmortem time of controlled freezing point (P < 0.05). * represents the significant
difference between the controlled freezing point and refrigeration at the same time of storage
(P < 0.05). Translated from Zhang et al. (2016)
shown in Fig. 12.7a, b. The gray scale values of the electropherograms in Fig. 12.7
were analyzed, and the overall phosphorylation level of different storage treatment
groups was shown in Fig. 12.8. The global phosphorylation level of myofibrillar
protein in postmortem lamb increased first and then decreased in ice temperature
storage group and cold storage group, the maximum phosphorylation level of the
control group was reached at day 3 after postmortem, and the CFPS group reached
the maximum phosphorylation level at 12 h postmortem. The myofibrillar protein
total level of phosphorylation in the control group was significantly higher than that
in the CFPS group, from the 24 h storage period to the end of the storage period
(P < 0.05).
Glycolysis is the most important mode of energy supply for postmortem muscles.
It is directly related to meat color, pH, water holding capacity (WHC), and muscle
rigor mortis. Meanwhile, sarcoplasmic protein accounts for more than two-thirds of
glycolytic enzymes (Mikkelsen et al. 1999). The large consumption of ATP in
postmortem muscles leads to an accelerated rate of glycolysis and a rapid decrease
in pH, which will accelerate the appearance of rigor mortis, promote aging, and
reduce the total time of rigor mortis (Woods et al. 2005). Other studies have shown
that protein phosphorylation plays an important role in regulating glycolytic enzyme
activity (Scopes and Stoter 1982). Therefore, the sarcoplasmic protein of postmor-
tem mutton is selected for detecting and analyzing its changes of phosphorylation
level under different storage conditions for six time points.
The global phosphorylation level of sarcoplasmic protein in the refrigerated
group was higher than that in the CFPS group at 12 h to day 3, and the CFPS
group was higher than the refrigerated group on the day 9 (Fig. 12.8). The reason
might be that the activity of protein kinase in the early stage of refrigerated storage
was higher than that in the CFPS group (Fig. 12.6), and the degree of phosphory-
lation of sarcoplasmic protein was stronger, with the prolongation of storage time,
the rate of glycolysis was faster in the control group (Li et al. 2016). ATP consump-
tion was faster and the content was decreased, resulting in decreased ATP-dependent
protein kinase activity (Dickens and Lyon 1995), and the level of reaction of
phosphorylation of sarcoplasmic protein was decreased, and it is no longer rising.
On the contrary, CFPS was beneficial for maintaining protein kinase activity,
thereby promoting the stabilization of the global phosphorylation level of sarcoplas-
mic proteins. Even though the refrigerated group was good for the global phosphor-
ylation level of sarcoplasmic proteins, not all refrigerated treatment had improved
the phosphorylation level of sarcoplasmic protein than CFPS. The global phosphor-
ylation level of sarcoplasmic protein was affected by the storage conditions, storage
time, and the interaction between storage conditions and storage time.
12.2.4 Effect of Controlled Freezing Point Storage

on Phosphorylation Levels of Myofibrillar Protein
The SDS-PAGE electropherogram of myofibrillar proteins in the CFPS group and

refrigerated group at 0.5 h, 12 h, 24 h, 3 d, 5 d, and 9 d after postmortem was shown
in Fig. 12.9a, b. Figure 12.9a showed the phosphorylated protein stained by Pro-Q
Diamond, and Fig. 12.9b showed the whole myofibrillar protein stained with
SYPRO Ruby. The gray scale values of the electropherograms in Fig. 12.9 were
analyzed, and the global phosphorylation level of the different storage treatment
groups was shown in Fig. 12.10. The global phosphorylation level of myofibrillar
Fig. 12.9 Gel images of phosphoproteins and total proteins of myofibrillar protein at different
storage temperature separated by SDS-PAGE. (a) Image of phosphoproteins stained with Pro-Q
Diamond. (b) Image of total proteins stained with SYPRO Ruby. Translated from Zhang et al.
(2016)
Fig. 12.10 The global phosphorylation level of myofibrillar protein at different storage tempera-
ture. The lowercases represent the significant difference between different postmortem time of
refrigeration (P < 0.05), while the capital letters represent the significant difference between
different postmortem time of controlled freezing point (P < 0.05). * represents the significant
difference between the controlled freezing point and refrigeration at the same time of storage
(P < 0.05). Translated from Zhang et al. (2016)
protein in postmortem lamb increased first and then decreased in two groups, and the
maximum phosphorylation level of the control group was reached at 24 h after
postmortem, and the CFPS group reached the maximum phosphorylation level at
12 h, and from the 24 h to the end of the storage period, the global phosphorylation
level of myofibrillar protein of the control group was higher than the CFPS group
(P < 0.05).
Myofibrillar protein is the most abundant protein in skeletal muscle, accounting

for 55–60% of the total protein. Myofibrillar protein is associated with muscle
contractile properties, and also strongly related to muscle functional properties and
raw meat cooking characteristics (Lee et al. 2010). Previous studies have found out
that a variety of myofibrillar proteins, such as myosin, troponin, actin, tropomyosin,
and myosin light chain, can be phosphorylated (Mazzei and Kuo 1984; Ryder et al.
2007; Toyo-Oka 1982). Combined with the mass spectrometry results from our team
(Chen et al. 2016) and Huang et al. (2012), this experiment chose to analyze the
phosphorylation level of some important myofibrillar protein bands.
12.3 Low-Variable Temperature and High Humidity

Thawing
According to the physicochemical properties and heat exchange characteristics of

ice-water during the thawing processes of frozen meat, the frozen meat is thawed at
2–6 C. Since the density of water is the highest at 4 C, the temperature fluctuates
around 4 C can change the density of water and promote the migration of water,
thus accelerating heat exchange and increasing the thawing speed. At the same time,
combined with high humidity thawing conditions, it protects the hydrated surface of
the frozen meat protein and inhibits the oxidation of meat during thawing. As
presented in Fig. 12.11, the LVTHHT improved based on the 4 C thawing storage
usually used in production. It fluctuates around 4 C, which has a low requirement
of refrigeration equipment and a low energy consumption. The effects of a LVTHHT
test group, thawing temperature 2 C ! 6 C ! 2 C, RH > 90%), and a
conventional air thawing method (control group, thawing temperature 4 C) on the
quality of lamb hindquarters were investigated. The indexes including color,
cooking loss, thawing loss, protein content of thawing drip, texture profiles analysis
(TPA) of thawed hindquarter, and surface hydrophobicity of myofibrillar protein
were measured. The microstructures of the transverse section of frozen and thawed
samples were observed by scan electric microscopy (SEM). The effects of different
thawing methods on the component, aggregation, and degradation of myofibrillar
protein were studied by SDS-PAGE gel electrophoresis (Fig. 12.12).

Humidity Thawing on Meat Quality Color, Thawing
Loss and Cooking Loss, Texture
Although the meat color has no significant influence on the nutritional value and
flavor of meat, it will affect consumers’ preferences. And it is also an apparent
manifestation of changes in muscle biology, biochemistry, and physiology, and an
12.3 Low-Variable Temperature and High Humidity Thawing 283
Fig. 12.11 Flow chart of thawing method of low-variable temperature with high humidity.
1-pressure gauge, 2-frozen meat, 3-humidity sensor, 4-steam value, 5-steam pipe, 6-temperature
sensor, 7-frequency refrigeration fan, 8-control system. Translated from Zhang et al. (2013)
important indicator of the meat sensory quality. From Table 12.1, we can see that the
L* value and a* values of the experimental group were both higher than those of the
control group. At 48 h, the L* value of the experimental group was 26.90, signifi-
cantly higher than that of the control group (P < 0.05), and the a* value was 10.56,
which was significantly higher than that of the control group (8.20) (P < 0.05).
There was no significant difference in the brightness value b* between the two
methods of thawing mutton. L* value represents the brightness value of the meat
sample, the larger the value, the better the gloss of the meat; a* value represents the
redness of the meat sample, the higher the value, the better the color of the meat, the
fresher the meat sample; b* value represents the yellowness of the meat, the higher
the value, the less fresh the meat (Xia et al. 2009). Thawing will reduce the freshness
of the meat color, but the experimental group had a fresher color than the control
group. In the thawing process, L* and a* values of both the experimental group and
the control group showed a trend of increasing at first and then decreasing, which
may be due to the presence of ice crystals on the surface of frozen meat samples in
the early thawing stage. The melting of ice crystals and the formation of
oxymyoglobin by myoglobin combining with oxygen can result in an increase in
brightness and redness values. In the late stage of thawing, the gloss loss occurs due
to excessive water loss of the meat sample that can cause a decrease in the brightness
value. The increase of the contact time between the meat sample and the air can be
the reason to explain the significant decrease in the redness value due to the increase
in the oxidation rate of myoglobin. It also reported that the oxidation and degradation
of myoglobin during thawing would lead to the deterioration of meat color (Xia et al.
2012).
284
Table 12.1 Effects of different thawing methods on lamp chromatic aberration. Translated from Zhang et al. (2013)
Control group Test group
12
Thawing time/h L* a* b* L* a* b*
0 27.32 0.48a 13.16 1.03a 6.84 0.11a 27.39 0.56a 13.06 0.74a 6.86 0.51a
12 34.57 2.8a 15.17 1.46b 7.72 0.89a 34.72 1.22a 18.39 1.89a 7.36 0.84a
24 36.67 0.51b 16.49 1.33b 7.42 1.41a 38.69 1.64a 19.01 1.05a 7.98 1.81a
36 27.35 2.08b 11.10 1.42b 6.81 0.70a 28.36 0.19a 13.43 1.82a 6.94 1.02a
48 24.83 1.03b 8.20 1.03b 5.76 0.55a 26.90 0.96a 10.56 0.96a 5.87 1.40a
Different letters in the same column indicate significant differences between the test group and control group (P < 0.05)
Improvement of Meat Quality by Novel Technology
Table 12.2 Effects of different thawing methods on thawing loss and cooking loss. Translated
from Zhang et al. (2013)
Index Thawing loss/% Protein content of drip/% Cooking loss/%
Control group 7.01 0.06a 13.00 0.04a 39.56 0.01a
Test group 3.01 0.03b 4.02 0.04b 35.97 0.02b
Thawing time was 48 h. Different letters in the same column indicate significant differences
between the test group and control group (P < 0.05)
Table 12.3 Effects of different thawing methods on texture of lamb. Translated from Zhang et al.
(2013)
Treatment Hardness/N Springiness Gumminess Chewiness/N
Control group 45.8 7.3b 0.62 0.05a 0.59 0.05a 16.6 2.4b
Test group 55.5 13.0a 0.70 0.06a 0.58 0.06a 22.7 3.9a
In the thawing process, frozen meat will lose juice, which contains a large amount
of soluble protein, resulting in nutrient loss of thawed meat. Compared with the
control group, the thawing loss rate of the experimental group was 3.01%, signifi-
cantly lower than that in the control group (7.01%, P < 0.05). The protein content in
juice was 4.02%, significantly lower than 13.00% in control group (P < 0.05).
The cooking loss was 35.97%, which was also significantly lower than the 39.56%
of the control group (P < 0.05) (Table 12.2). The results showed that, compared with
the conventional air thawing, LVTHHT could significantly reduce the nutrient loss
and improve the quality of thawed mutton.
Many domestic and international scholars have reported that TPA can be used to
evaluate the texture characteristics of meat and meat products (Dai et al. 2008;
Johnson et al. 2010; Martinez et al. 2004; Xia et al. 2012). In this experiment, the
texture characteristics of thawed mutton under different thawing conditions were
analyzed (Table 12.3). The results showed that the hardness and chewiness of the
experimental group were 55.5 and 22.7 N, respectively, which were significantly
higher than 45.8 and 16.6 N of the control group (P < 0.05), but there were no
significant differences in the elasticity and cohesion of the two groups. Chewability
is a comprehensive manifestation of hardness, elasticity, and cohesiveness, reflecting
the energy required for lamb meat from chewable state to swallowing state. Within a
certain range, the greater the value is, the better the corresponding “bite sensation” of
meat taste will be (Dai et al. 2008; Ruan et al. 2008). Table 12.3 showed that the
chewiness of the thawed mutton in the experimental group was significantly better
than that in the control group (P < 0.05), which indicated that the LVTHHT
significantly improved the texture characteristics of the thawed mutton compared
with the conventional air thawing method.

Humidity Thawing on Myofibril Protein
Protein surface hydrophobicity is an indicator of protein hydration ability. The larger

the value, the smaller the protein hydrophobicity, the weaker the ability to bind with
water, the higher the rate of thawing loss. The results showed that the hydrophobicity
of proteins increased by two different thawing methods, but the degree of increase
was different. For example, the hydrophobicity of the mutton was 213.32 after 48 h
of thawing in the control group, which was nearly 5 times higher than the hydro-
phobicity of the unthawed mutton 44.08, while the hydrophobicity of mutton before
and after thawing in the test group was only more than 2 times higher (Table 12.4).
Therefore, compared with the conventional air thawing method, the LVTHHT
method can delay the reduction of hydration capacity of protein during the thawing
process, thus improving the WHC of thawed meat.
Meat proteins can be divided into water-soluble sarcoplasmic proteins, saline-
soluble myofibril proteins, and insoluble matrix proteins according to their solubil-
ity, and these three types of protein have different physical properties (Ruan et al.
2008). Wagner and Añon (2007) believed that denaturation of salt-soluble protein
had the greatest influence on meat quality. In this experiment, SDS-PAGE electro-
phoresis was used to observe the changes in the composition of the protein of the
salt-soluble myofibrillar protein after thawing (Fig. 12.12). As shown in Fig. 12.12,
there were 5 major bands of lamb myofibril, and from top to bottom they were:
myosin heavy chain (MHC) with molecular weight of 220 kDa, paramyosin with
molecular weight of 100 kDa, 43 kDa actin, 36 kDa tropomyosin, and 35 kDa
myosin (troponin T) subunit (protoplasts binding subunit). The results of this study
were consistent with those reported by Thanonkaew et al. (2006). The SDS-PAGE
profiles of unfrozen myofibrillar protein (1,2) were compared with those of air
thawing (3,4) and LVTHHT meat (5,6). The comparative analysis showed that
thawing resulted in the protein denaturation, degradation and enve new cross-
linking, and the thawed mutton electrophoresis map produced new protein bands
at about 55 kDa and 30 kDa, and the disappearance of protein bands at about 40 kDa.
Similar cases existed in the protein band below 29 kDa. This may be due to the
oxidation of myofibril during thawing, which will lead to protein cross-linking and
degradation (Park et al. 2007; Xia et al. 2012).
Table 12.4 Surface hydrophobicity of mutton protein during thawing. Translated from Zhang et al.
(2013)
Surface hydrophobicity
Treatment 0 24 h 48 h
Control group 44.08 2.31a 125.68 4.64a 213.32 5.26a
Test group 44.14 3.21a 72.67 6.77b 115.88 13.55b
Fig. 12.12 Effects of different thawing methods on SDS-PAGE of myofibril protein. M is standard
protein; 1, 2 are non-thawing meat protein; 1, 4 are thawing meat protein in natural condition (in air
at 4 C) for 48 h; 5, 6 are thawing meat protein in low-variable temperature with high humidity for
48 h. Translated from Zhang et al. (2013)

Humidity Thawing on the Microstructure of Meat
The frozen lamb and lamb thawed by two different methods were observed by
scanning electron microscopy to study the integrity of the muscle fiber bundle, the
interfiber bundle space and the integrity of the muscle bundle membrane. The results
were shown in Fig. 12.13 (magnification 500 times), which showed that the
unthawed mutton muscle fiber bundles were parallel to each other, the muscle
fiber bundles were closely arranged with small interspaces, and the structure of the
perimysium was intact (Figure 12.13a). And thawing can lead to the loss of the
integrity of the muscle fiber structure, the increase of the gap between the muscle
bundles, the destruction of the dense structure, and the rupture of the myocutaneous
membrane. Previous researches have studied the physicochemical changes of the
pig muscle Longissimus thoracis and the effect on protein oxidation under repeated
freezing and thawing conditions (Xia et al. 2012). It was also discovered that freeze-
thaw treatment could destroy the microstructure of muscle and increase the gap
between muscle fibers. However, the muscle fiber structure of the experimental
group was more complete than that of the control group, the gap was smaller than
Fig. 12.13 Effects of different thawing methods on fiber bundle of mutton for thawing 48 h
(500). Translated from Zhang et al. (2013)
that of the control group, and the muscle bundle membrane was damaged to a lesser
extent (Figure 12.13b, c). Muscle fascia has a certain elasticity, which plays a role in
maintaining muscle integrity and preventing muscle fiber damage. The destruction
of the fascicular membrane will lead to the loss of muscle tissue integrity, the
destruction of the dense structure, the enlargement of the space between the muscle
fiber bundles, and the decrease of the WHC of the muscle, resulting in more water
leakage and serious juice loss. Compared with air thawing, LVTHHT has less
damage to the texture of thawed mutton, and the muscle bundle structure of the
meat was more complete and dense, thus better WHC, lower juice loss rate, and
better chewiness, which also verified the results of this study on the juice loss rate
and texture change of thawed mutton.
12.4 Pulse Pressure Pickling
In this part, mutton was used as the research object, and discussed the effects of pulse
pressure salting and atmospheric pickling (as shown in Fig. 12.14) on meat quality.
Pulse pressure pickling machine (self-developed): this experimental device sim-
ulates the pulse pressure salting process. The material is designed to be placed in the
body of the pickling chamber (marked 1 in Fig. 12.15). The pressure regulating
device is used to realize the pulse pressure salting process.
12.4.1 Effect of Pulse Pressure Salting on Meat Quality
12.4.1.1 Salt Content of Lamb
As shown in Fig. 12.16, the salt content in the mutton increased with the pickling
time, and the pickling efficiency of pulse pressure salting was improved 8–26% than
atmospheric pickling. The main reason was that during the pulse pressure salting
12.4 Pulse Pressure Pickling 289
Salting liquid
Gas out NaCl increase ˈ Water
+ ü Na+
Na+Clü Na Cl
Clü
Step 1˖atmospheric Step 2˖vacuum Step 3˖return to

pressure phase atmospheric pressure
Fig. 12.14 The scheme of the steps in pulsed vacuum pickling. Translated from Xu (2014)
Fig. 12.15 Schematic diagram of pickling machine with pulsed pressure. 1-Salting cavity,
2-Sealing cover, 3-Curing tank and control device, 4-Air inlet, 5-Control valve, 6-Pressure regula-
tor, 7-Drain valve. Translated from Xu (2014)
process, the pressure amplitude changed continuously between vacuum and atmo-
spheric pressure, and the meat structure changed according to the fluid dynamics
principle and the deformation relaxation phenomenon, effectively promoting the
pickling liquid flow within the meat tissue (Ertekin and Cakaloz, 1996). Compared
with the group of atmospheric pickling, when the pulse pressure salting was in a
vacuum state, the structure of the meat tissue expanded, and the internal gas and the
free state water were discharged, which hindered the entry of the curing liquid. When
the pressure returned to normal pressure, the pickling liquid quickly entered the
inside of the meat pores, thereby accelerating the migration of the solute material
(Dong et al. 2008; Xu et al. 2010; Paes et al. 2006), which promoted the uniform
distribution of the salt (Gonz Lez-Mart Nez et al. 2002; Wang et al. 2013).
5.0
4.5 脉ࣘ真空腌制
Pulsed vacuum salting
4.0 常঻腌制
Atmospheric salting
3.5
Salt content /%
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.0 1.5 3.0 4.5 6.0
Salting time /h
Fig. 12.16 Effect of different brining methods on salt content of lamb. Translated from Xu (2014)
75 脉ࣘ真空腌制
常঻腌制
Atmospheric salting
73
Water content /%
71
69
67
65
63
0.0 1.5 3.0 4.5 6.0
Salting time /h
Fig. 12.17 Effect of different brining methods on water content of lamb. Translated from Xu
(2014)
12.4.1.2 Water Content of Lamb
It is shown in Fig. 12.17 that the water content of lamb was decreasing during the
pulse pressure salting process, and after pickling was significantly lower than before
pickling (P < 0.05). During the 1.5 h period before pulse pressure salting, the water
decreased rapidly and reached equilibrium at around 4.5 h. During the atmospheric
pickling process, the water content decreased first and then increased, and the water
content in the lamb after pickling was not significantly different from that before
pickling (P > 0.05). After marinating by two kinds of pickling methods, the water
content of lamb after pulsating vacuum pickling was significantly lower than that of
atmospheric pickling (P < 0.05). The change of water content of lamb during
atmospheric pickling was mainly due to the fact that the sodium chloride pickling
solution was at stationary statement, causing denaturation and dehydration of the
external protein of the lamb, with the advancement of curing, sodium chloride
gradually migrates into the interior of the lamb and formed a complex with the
protein. This salt complex had a greater driving force, resulting in a small positive
increase in water content, and there was no significant change after 4.5 h (P < 0.05).
The pulsating pressure during the pulse pressure salting process is the main control
force of free water in the lamb.
12.4.1.3 Color of Lamb’s Surface
It is shown in Table 12.5 that the two pickling methods had a significant effect on the
surface color of lamb, and the color indicators L*, a*, b* were significantly lower
than the fresh meat. The L* value represents the brightness of the color, a* represents
the redness value, and b* represents the yellowness value (Amiryousefi et al. 2014).
The decrease of L* value may be due to changes in the water state and content of the
lamb after curing, and the water dispersed in the muscle fibers may affect the color
reflectance of the meat (Shi et al. 2011). The redness value decreased (P < 0.01),
mainly due to the lamb in the marinade, and one was the hemoglobin dissolution in
the meat, the other was that the marinade directly contacted the lamb tissue to
promote the conversion of myoglobin to high-iron myoglobin and its derivatives.
Accompanied by the degeneration of myoglobin (Brewer and Novakofski, 1999;
King and Whyte, 2006; Villamonte et al. 2013; Yancey et al. 2011), it made its color
lighter. In the pickling process, sodium chloride in the marinade will aggravate the
oxidation of fat in the meat, which is one of the causes of the decrease in the
yellowness value (Bello and Granados, 1996).
As shown in Table 12.6, the effects of the two pickling methods on the internal
color of the lamb were different. The a* value was significantly lower than that of
fresh meat after pulsating pickling, but there was no significant difference after
Table 12.5 Effect of different brining methods on color of lamb’ surface Translated from Xu
(2014)
salting time /h
index pickling method 0 6 saliency
L* Pulse pressure salting 40.56 1.35 37.52 1.41 **
Atmospheric pickling 40.56 1.35 36.82 2.08 **
a* Pulse pressure salting 18.40 0.76 4.50 0.62 **
b* Pulse pressure salting 12.03 0.40 4.37 1.07 **
**Very significant difference (P < 0.01)
Table 12.6 Effect of different brining methods on color of lamb’s interface Translated from Xu
(2014)
salting time/h
index pickling method 0 6 saliency
L* Pulse pressure salting 40.06 1.53 34.33 2.12 **
a* Pulse pressure salting 18.36 0.24 15.86 0.52 **
Atmospheric pickling 18.36 0.24 18.3 2.85
b* Pulse pressure salting 4.91 0.84 4.33 0.51
Atmospheric pickling 4.91 0.84 4.30 0.72
*Significant difference(P < 0.05), **very significant difference (P < 0.01)
6.1 脉ࣘ真空腌制
6.0 常঻腌制
Atmospheric salting
5.9
pH
5.8
5.7
5.6
5.5
0.0 1.5 3.0 4.5 6.0
Salting time /h
Fig. 12.18 Effect of different brining methods on pH of lamb. Translated from Xu (2014)
atmospheric pickling. The change of the internal color of the lamb was mainly due to
the migration of sodium chloride in the marinade. Sodium chloride can promote the
conversion of myoglobin to high-iron myoglobin and make the color of the meat
brown (Corzo et al. 2006a). Pulse pressure salting can effectively promote the
internal penetration of sodium chloride to make its internal salt content significantly
higher than that of atmospheric pressure pickling. It was the main reason for the
internal color index a* of the meat sample after pulse pressure salting was signifi-
cantly lower than 0 h and atmospheric pressure (P < 0.05).
12.4.1.4 pH
The change of pH of lamb during pickling was shown in Fig. 12.18. It can be seen
that there were some differences in the effects of different pickling methods on the
change of pH of lamb. In the 3 h stage before pulse pressure salting, the pH of lamb
showed an upward trend, and there was no significant change after 3 h (P > 0.05).
During the atmospheric pickling process, the pH of lamb increased first and then
decreased, and the overall pH was lower than that of the pulsating pickling process.
When the curing was finished, the pH of lamb in pulse pressure salting was
significantly higher than that of in atmospheric pickling (P < 0.01). The main reason
for the decrease in the pH of the atmospheric pickling was that as the pickling time
prolonged, the liquid in the raw material tissue gradually oozed out due to the
influence of the osmotic pressure, resulting in a decrease of pH (Zhang and Xiong,
2013). The pH of lamb during the pulse pressure salting process was mainly because
of the action of the pressure during the curing process, which promoted the flow of
the marinade in the lamb and accelerated the exudation of the internal gas and free
tissue fluid of the lamb. The sodium chloride pickling solution was neutral and the
pH rose.
12.4.1.5 Cooking Loss and Water Holding Capacity
The water holding capacity of meat, also known as water power or WHC, refers to
the ability of muscles to retain and gain water when subjected to external forces (Yin,
2011). Generally, the indexes to measure the hydraulic retention of muscle include
WHC, drip loss, cooking loss, centrifugal loss, purge loss, etc. (Kaale et al. 2014;
Sánchez-Valencia et al. 2014; Zhou et al. 2014). Two indicators of cooking loss and
WHC were selected for this experiment. As shown in Fig. 12.19, the cooking loss of
lamb after pickling decreased significantly (P < 0.05), but there was no significant
difference between the two pickling methods (P > 0.01). As shown in Fig. 12.20, the
WHC of lamb increased significantly (P < 0.05) before 1.5 h during the pulse
pressure salting, and it was stable during the 1.5–6 h (P > 0.01). The increase in
50
45
Atmospheric salting
40
Cooking loss /%
35
30
25
20
15
10
0.0 1.5 3.0 4.5 6.0
Salting time /h
Fig. 12.19 Effect of different brining methods on cooking loss of lamb. Translated from Xu (2014)

100
Atmospheric salting
90
80
WHC/%
70
60
50
40
30
0.0 1.5 3.0 4.5 6.0
Salting time /h
Fig. 12.20 Effect of different brining methods on WHC of lamb. Translated from Xu (2014)
WHC was because when sodium chloride acted directly on muscle fibers, the ionic
strength was >0.15, and the muscle fibers would have an electrostatic shielding
effect due to the combination of salt ions, resulting in swelling of the muscle fibers,
an increase in area, and an increase in WHC (Thorarinsdottir et al. 2011b), and
reached a maximum at an ionic strength of 0.8.
The ionic strength of the marinade selected in the experiment was close to 2.56,
and the final salt content in the meat sample after curing was 1.5%–2.5% (ionic
strength 0.25–0.42), that is, the ionic strength was just in the range of swelling of the
muscle fiber. At this time, a large amount of chloride ions were trapped between the
myofibrils, increasing the electrostatic repulsion caused by the negative charge, thus
leading to the myofibril to swell and increasing the WHC, which was similar to that
of Thorarinsdottir reported in the early stage of squid pickling (Thorarinsdottir et al.
2011a). Meanwhile, as the curing time increased, the sarcoplasmic protein and
myofibrillar protein were dissolved (Chaijan, 2011). In the case of heat denaturation,
the water and fat were wrapped and solidified, and the WHC increased.
12.4.1.6 Tenderness
As shown in Fig. 12.21, with the extension of pickling time, the shear force of lamb
presented a downward trend. The shearing force of lamb was significantly decreased
(P < 0.05) before 1.5 h during pulse pressure salting, and there was no significant
change after 1.5 h (P > 0.01). The shear force of lamb during pulse pressure salting
was lower than the atmospheric pickling during pickling. The shear force is an
important indicator for the meat tenderness. The higher the shear force value,
the firm the meat is, and vice versa (Devine et al. 2002; Karumendu et al. 2009).
The improvement of meat tenderness may be due to the swelling of muscle fibers,
80
70
Atmospheric salting
60
Shrae force /N
50
40
30
20
10
0
0 1.5 3 4.5 6
Salting time /h
Fig. 12.21 Effect of different brining methods on Warner–Bratzler shear force of lamb. Translated
from Xu (2014)
the dissociation of myosin, the increase in WHC, and the increase in tenderness at
high ion concentrations. On the other hand, during the pickling process, because of
the pulse action, the lamb has a dynamic change of expansion-contraction, which
causes the lamb tissue to relax, thereby improving the tenderness (Tobin et al. 2013).
It has been reported that pulse pressure salting causes the pickling fluid to flow in the
meat due to the hydrodynamic action and the deformation relaxation mechanics due
to the cyclical changes in pressure. The mechanical force generated by the flow of
the pickling liquid is a kind of massage for lamb, and is also one of the reasons for
promoting salt migration and improving tenderness (Koutchma, 2007).
12.4.2 Effect of Pulse Pressure Salting on Myofibrillar

and Sarcoplasmic Protein
The results of SDS-PAGE of myofibrillar protein of lamb were shown in Fig. 12.22,
as can be seen, the MHC (220 kDa), actin (actin,43 kDa), paramysin
(paramysin,100 kDa), and other bands of myofibrillar protein were clearly visible,
while tropomyosin was near 36 kDa, and myogenic protein was near 35 kDa, which
was consistent with the previous literature (Zhang and Xiong, 2013). The disap-
pearance of the band or the formation of a new band was not observed in Fig. 12.22
compared to 0 h before pickling, the color of electrophoretic pattern of pulse pressure
salting and atmospheric pickling became thicker at the bands of the MHC and actin,
which was due to the dissolution of myosin and actin in the mutton tissue during the
curing process. At the band of 40 kDa, the color of the band gradually darkened as
the pickling time increased, which might due to dissociation of the tropomyosin
protein.
kDa M 0 1 2 3 4 5 6 7 8
myosin heavy chain
25
paramysin
150
100
70
actin
50
40 40 kDa
36 kDa
35 kDa
30
20
Fig. 12.22 Effects of different pickling methods on SDS-PAGE of myofibril protein. M is standard
protein; 0 is fresh lamb protein; 1 ~ 4 are pulsed vacuum pickling meat protein with 1.5, 3, 4.5, 6 h;
5 ~ 8 are atmospheric pressure pickling with 1.5, 3, 4.5, 6 h. Translated from Xu (2014)
kDa M 0 1 2 3 4 5 6 7 8
390 kDa
250
150 151 kDa
100
70
50
40 39 KDa
Fig. 12.23 Effects of different pickling methods on SDS-PAGE of sarcoplasmic protein. M is

standard protein; 0 is fresh lamb protein;1 ~ 4 are pulsed vacuum pickling meat protein with 1.5,
3, 4.5, 6 h; 5 ~ 8 are atmospheric pressure pickling with 1.5, 3, 4.5, 6 h. Translated from Xu (2014)
Figure 12.23 showed the SDS-PAGE electrophoresis of the sarcoplasmic protein

of lamb. The macromolecular bands were generated at 390 kDa; the color of the
band at 150 kDa increased with the pickling time; the band at 39 kDa was pulsating.
After pickling for 4.5 h, it became lighter, but the change in atmospheric pickling
was not obvious.
12.4.3 Effect of Pulse Pressure Salting on Microstructure

of Meat
From Fig. 12.24 we could tell that the ultrastructure of lamb has changed after pulse
pressure salting. Figure 12.24a shows the raw meat that has not been marinated after
thawing. The myofibrils were neat and clearly visible, with regular light and dark
stripes, and M line, Z line, A band (dark band), I band (The bright band), and the H
zone (the brighter strip in the middle of the dark band) were clearly visible. After
1.5 h of pulse pressure salting, the muscle fibers began to shrink and the Z line began
to disappear. After 3 h, the muscle fibers expanded and became thicker. After 6 h of
pulse pressure salting, the Z line of the lamb was thinner and fuzzier than the raw
meat.
Fig. 12.24 The longitudinal section of lamb muscle at transmission electron microscope under
pulsed vacuum pressure. (a) Lamb muscle at 0 h; (b) lamb muscle under pulsed vacuum pressure for
1.5 h; (c) lamb muscle under pulsed vacuum pressure for 3 h; (d) lamb muscle under pulsed vacuum
pressure for 4.5 h; (e) lamb muscle under pulsed vacuum pressure for 6 h. Translated from Xu
(2014)
Fig. 12.25 The longitudinal section of lamb muscle at transmission electron microscope under
atmospheric pressure. (a) Lamb muscle at 0 h; (b) lamb muscle under atmospheric pressure for 1.5
h; (c) lamb muscle under atmospheric pressure for 3 h; (d) lamb muscle under atmospheric pressure
for 4.5 h; (e) lamb muscle under atmospheric pressure for 6 h. Translated from Xu (2014)
As shown in Fig. 12.25, the atmospheric pickling process has changed the
ultrastructure of lamb. After 1.5 h of pickling, the muscle fibers began to shrink
and the gap gradually decreased; the Z line became shallower and gradually
disappeared; after 6 h of pickling, it can be seen from the figure that the H band of
the lamb tended to be blurred.
Compared with pulse pressure salting, the Z-line of lamb tended to be lighter and
will be thinner and more blurred after pulse pressure salting, the main component of
the Z-line was actin. It indicated that both kinds of pickling methods dispelled the
actin of lamb, and the pulse pressure salting caused the dissociation of actin to be
12.5 Conclusions 299
2.2 Pulsed vacuum salting

Atmospheric salting
Sarcomere length (μm) 2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.0 1.5 3.0 4.5 6.0
Salting time (h)
Fig. 12.26 Effect of different brining methods on sarcomere length of lamb. Translated from Xu
(2014)
more than that of normal pressure pickling. This was consistent with the results of
the effect of pulse pressure salting on myofibrillar protein in Fig. 12.22.
In terms of Fig. 12.26, during the pickling process, the length of the sarcomere of
lamb decreased. The length of sarcomere was not significantly changed after pulse
pressure salting (P > 0.05). The length of sarcomere was significantly lower than
that of raw meat after atmospheric pickling (P < 0.05), and the length of the
sarcomere after pulse pressure salting was significantly higher than the length of
the sarcomere after atmospheric pickling (P < 0.05). In the present study, the ionic
strength is just in the range of muscle fiber expansion, a large amount of chloride
ions are trapped between the myofibrils, increasing the electrostatic repulsion caused
by the negative charge, causing the expansion of the myofibrils, which is the reason
for the increase in WHC. At the same time, the dissolution of sarcoplasmic protein
and myofibrillar protein with the increase of pickling time was one of the reasons for
myofibrillar structure change during the pickling process. There was no significant
change in the length of the sarcomere after pulse pressure salting. It might be that
during the pulse pressure salting process, due to the periodic vacuum pulsation
process, the vacuum pressure exerted an outward expansion force on the lamb.
However, the length of the sarcomere was shortened because of pickling, and the
combined effect of the length of the sarcomere was not significantly changed after
pulse pressure salting.
12.5 Conclusions
Fresh ovine meat stored in CFPS treatment for 10 days proved to have better color
stability in comparison with those in 4 C storage. CFPS could be used as an option
storage method for color improvement of fresh meat. The samples in this study were
over wrapped by polyvinyl chloride film during storage and the effects of different
packaging conditions could be investigated in the next study as packaging could
affect potential effects of storage procedure by interacting with storage treatments.
Compared to the most common thawing method (in air at 4 C), LVTHHT
method had many advantages: it prevented the loss of the surface water, significantly
reduced thawing loss, cooking loss, and nutrition loss (protein). Moreover, the
formation of water film prevented meat oxidation and delayed the deterioration of
meat quality; therefore, the physicochemical characteristics of the thawed meat were
closer to fresh meat. LVTHHT method could be used for thawing frozen meat.
Compared to atmospheric pressure pickling, the salt content of lamb with PVP
was improved by 8–26%. The cooking loss and Warner–Bratzler shear force
decreased notably, and WHC increased significantly. Therefore, the PVP could be
used to improve the curing efficiency while maintaining the quality of lamb.
Acknowledgments Parts of this chapter are translated from Society of Agricultural Engineering,
29, Zhang, C. H., et al. Low-variable temperature and high humidity thawing improves lamb quality
(Chinese). 267–273; Scientia Agricultura Sinica, 49, Zhang, Y., et al. Effects of controlled freezing
point storage on the protein phosphorylation level (Chinese). 4429–4440. Copyright (2020), with
permission from two Journals. Xu, W. 2014. Optimization of pulsed vacuum pressure pickling and
its effect on the quality of lamb. Ningxia University. Zhang, Y. 2016. Study of controlled freezing
point storage regulating lamb color stability and its protein phosphorylation level. Shaanxi Normal
University.
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Dequan Zhang · Xin Li · Li Chen

Chengli Hou · Zhenyu Wang

Li Chen Chengli Hou

ISBN 978-981-15-9440-3 ISBN 978-981-15-9441-0 (eBook)

© Springer Nature Singapore Pte Ltd. 2020

Beijing, China Dequan Zhang

Part I Relationship Between Protein Phosphorylation and Meat Quality

5 Protein Phosphorylation Affects Meat Water Holding Capacity . . . 77

Part II Mechanism of the Effect of Protein Phosphorylation

6.4.4 Glycolytic Enzymes Activities and Phosphorylation

8.2.4 The Degradation of μ-Calpain 80 kDa Subunit . . . . . . . 153

9.3.2 Changes of Myoglobin Relative Contents . . . . . . . . . . 198

Part III Improvement of Meat Quality by Regulating Protein

11.2.1 Global Phosphorylation of Myoﬁbrillar Proteins with

12.3.3 Effect of Low-Variable Temperature and High

1.1 Meat Quality and Mechanism from Muscle to Meat

1.1.1 Importance of Meat Quality

© Springer Nature Singapore Pte Ltd. 2020 1

1.1.2 Mechanism of Meat Quality Formation During

1.2 Protein Post-Translational Modiﬁcation on Meat

1.2.1 Deﬁnition and the Role of Protein Post-Translational

Post-translational modiﬁcations, referring to the covalent enzymatic modiﬁcations of

succinyl group changes lysine’s charge from +1 to 1 and introduces a relatively

1.2.2 Role of Protein Post-Translational Modiﬁcation

biomarkers to track the processing time. In postmortem muscles, ubiquitination,

1.3 Current Research of Protein Phosphorylation (Live

Among all post-translation modiﬁcations, protein phosphorylation is one of the most

postmortem rate of pH decline by regulating the phosphorylation of sarcoplasmic

Abstract With the advancement of technical approaches, signaling pathways have

Keywords Protein phosphorylation · Methods · 2DE-gel · Pro-Q Diamond · TiO2

Proteins can be modiﬁed through several modiﬁcations with several functional

© Springer Nature Singapore Pte Ltd. 2020 11

2.2 Principle of Phosphorylated Protein Detection

Analysis of the phosphorylated protein is necessary for interpreting the fundamental

by gel electrophoresis ( an immunoprecipitation step for a particular protein). Then

2.3 Gel-Based Phosphorylated Protein Detecting Methods

2.3.1 Separation of Proteins by 2DE Gel

Two-dimensional electrophoresis of proteins, ﬁrstly introduced by O’Farrell (1975)

2.3.2 Detection of Phosphorylated Protein by Pro-Q Diamond

2.3.3 Detection of Phosphorylated Protein by Phos-Tag

A dinuclear metal complex of 1, 3-bis[bis(pyridin-2-ylmethyl) amino] propan-2-

2.4 Detection of Phosphorylated Proteins by LC-MS/MS

2.4.1 Labeling Treatment of Samples

2.4.2 Label Free Treatment of Samples

2.4.3 Enrichment of Phosphorylated Peptides Using

and comparatively higher concentrations of triﬂuoroacetic acid, which reduced the

2.4.4 LC-MS/MS Analysis

Protein phosphorylation is an extraordinarily important component of various signal

Keywords Meat color · Protein phosphorylation · Sarcoplasmic proteins ·

Electronic Supplementary Material The online version of this chapter (https://doi.org/10.1007/

© Springer Nature Singapore Pte Ltd. 2020 23

3.2 Changes of Meat Color After Protein Phosphorylation

Protein phosphorylation in postmortem muscle played essential roles in meat qual-

3.2.1 Global Level of Protein Phosphorylation After

Gel-based analysis of sarcoplasmic protein phosphorylation was shown in Fig. 3.1

3.2.2 Color of Meat with Different Protein

3.2.3 Role of pH and Lactic Acid Content on Color of Meat

Phosphatase inhibition Saline control

Phosphatase inhibition Saline control

decline signiﬁcantly inﬂuenced protein characteristics in postmortem muscle, for

0.489 * 0.541* 0.556* 0.029 0.567* 0.179