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64 Current Protein and Peptide Science, 2014, 15, 64-74

The Colostrum Proteome, Ruminant Nutrition and Immunity: A Review

Lorenzo E. Hernández-Castellano1, Andrè M. Almeida2,3, Noemi Castro1,* and Anastasio Argüello1

1
Department of Animal Science, Universidad de Las Palmas de Gran Canaria, Arucas, Gran Canaria, Spain;
2
Instituto de Tecnología Química e Biologica, Universidade Nova de Lisboa, Oeiras, Portugal; 3Instituto de
Investigação Científica Tropical, Lisboa, Portugal

Abstract: In this review authors address colostrum proteins implications in different domestic ruminant species. The
colostrogenesis process and how different factors, such as litter size or nutrition during gestation can alter the different
components concentrations in colostrum are also reviewed. The different colostrum fractions will be described, focus-
ing on high and low abundant proteins. This review describes the major function of such proteins and their role on the
passive immune transfer and nutrition in the newborn animal. It will be also performed a comprehensive review on dif-
ferent techniques and commercial kits available for high abundant protein depletion in colostrum. We will finally focus
on how proteomics has been used to address this issue and how it can contribute to the major questions about colos-
trum associated immunology.
Keywords: Colostrum, depletion, immune system, low abundant proteins, proteomics.

1. INTRODUCTION low abundant proteins (LAP) that could be involved in im-

munological processes. These processes have a high rele-
The relation between colostrum and newborn ruminant
vance to animal survival as they increase the protection
survival has been deeply characterized [1-4]. Colostrum is
against infections in newborn ruminants.
the first source of nutrition in neonate ruminants, having a
fundamental biological function in these animals, promoting
2. TIME OF SECRETION AND IMPORTANCE OF
immunoglobulin transfer from the dam to the newborn and
COLOSTRUM IN DOMESTIC RUMINANT SPECIES
providing protection against infections in the newborn [5].
Moreover, it has been described that colostrum is a mixture As long described, the complexity of the epitheliochorial
of diverse components, such as fat, lactose, vitamins and (cows and water buffaloes; [16, 17]) or synepitheliochorial
minerals that have a high nutritional importance [6]. How- (small ruminants; [18]) placentas do not allow an adequate
ever, in spite of the nutritional function, colostrum contains a transfer of immunoglobulins (Ig’s) from the dam to the
complex mixture of proteins that actively participate in the foetus, as in other species such as humans or mice
protection of the neonate against pathogens and other post- [hemochorial placenta; 19]. For this reason, newborn rumi-
partum environmental challenges [7]. nants are considered agammaglobulinemic (calves) or hypo-
gammaglobulinemic (lambs and kids) at birth [1, 2, 4, 20].
Proteomics is the field of science that, on a large scale, As a consequence, colostrum intake and the absorption of
studies the proteome, i.e. the set of proteins being expressed colostrum proteins play an essential role in PIT and ulti-
in a given tissue, organ, organism or fluid. Proteomics is a mately on newborn survival rates [3, 14, 15]. Such protein
relatively novel field and one of the fastest-growing areas in absorption is mainly based on immunoglobulins (IgG, IgM
biological research, receiving international recognition and, and IgA, mainly), however it has been also described the
additionally, it’s applications in animal science are wide and importance of non-immunoglobulin proteins absorption from
diverse [8, 9], including for instance, the transformation of colostrum to the newborn ruminants blood in order to ensure
muscle to meat [10], or dairy production [11]. Concerning a correct PIT [21]. In spite of this primary role, colostrum is
colostrum, proteomics has been used for instance to charac- also important to the newborn animal as the first source of
terize protein changes in the transition from colostrum to milk nutrition, supplying essential nutrients such as fat, proteins,
in cattle [12-14] and also to determine proteolytic processes lactose and minerals at this early stage of life [6]. Finally,
in stomach and gut of piglets upon colostrum intake [15]. colostrum also contains other substances such as antimicro-
This review describes the important role of colostrum in bial and anti-inflammatory agents [14], as well as growth
the passive immune transfer (PIT), particularly the high factors that control early gastrointestinal development [22],
abundant proteins (HAP) present in colostrum from different cytokines, enzymes and numerous other peptides [23, 24] as
ruminant species. Moreover, it is also described in detail the shown in (Fig. 1). Many of the bioactive whey and milk fat
globule membrane (MFGM) proteins, notably immuno-
*Address correspondence to this author at the Department of Animal Scien-
globulins, lactoferrin and growth factors, are present in co-
ce, Universidad de Las Palmas de Gran Canaria, Arucas, Gran Canaria, lostrum in higher concentration than in milk, reflecting the
Spain; Tel: +34 928451093; Fax: +34 928451142; importance of colostrum in the health of newborn ruminants
E-mail: [email protected] [25, 26].

1875-5550/14 $58.00+.00 © 2014 Bentham Science Publishers

The Colostrum Proteome, Ruminant Nutrition and Immunity Current Protein and Peptide Science, 2014, Vol. 15, No. 1 65

Fig. (1). Schematic representation of the major known proteins involved in the host defence system in milk and colostrum. (Adapted from
Wheeler et al. [27]). MFGM: Milk Fat Globule Membrane.

The absorption of these colostrum components is fa- ingly, and in order to increase the coverage of the colostrum
voured by a low proteolytic activity in the gastrointestinal proteome, it is necessary to study the different components
tract of newborn animals [28] and also by trypsin inhibitors separately. Some of these techniques are based in the pre-
present in colostrum [29-31]. However, these special condi- fractionation of the colostrum sample in order to reduce
tions for intact colostrum proteins absorption decrease along complexity before proteomics analysis [45]. These previous
the first 48 hours after birth, so it is crucial to feed newborn steps are in accordance with the different properties of the
ruminants with colostrum during this period [32, 33] in order proteins, namely charge, size or hydrophobicity.
for them to acquire an adequate initial concentration of se- The gel-based approach has been the most used method
rum Ig’s [34, 35]. In addition, it has been described that for decades, and is still widely used, being the two-
newborn ruminants that are not fed with colostrum during dimensional gel electrophoresis (2DE) method commonly
the first hours of life are more susceptible to diseases, con- employed in quantitative proteomics [46]. However, it has
siderably increasing mortality rates [36-38]. For all these been also described a clear interest on liquid chromatography
reasons, colostrum intake is very important to ensure the separations [15], which have several distinct advantages in
survival of newborn ruminants. comparison with the 2DE studies. Finally, mass spectrometry
(MS) has increasingly become the method of choice for
3. PROTEOMICS AS A TOOL TO ANALYZE AND analysis of complex protein samples. MALDI TOF/TOF
IDENTIFY PROTEINS FROM COLOSTRUM MS, for instance is a robust, sensitive and relatively inexpen-
sive. Moreover, this method has produced much of the pro-
Colostrum is a complex body fluid produced by the
tein identifications data reported in the literature [47]. How-
mammary gland of pregnant mammals. Colostrum composi-
ever, there are other techniques, such as isobaric tag for rela-
tion is regulated by hormones (estradiol and progresterone)
tive and absolute quantitation (iTRAQ) that allows studying
during colostrogenesis [3] that may be defined as the prepar-
deeply the proteome of complex samples with higher accu-
tum transfer of components, mainly immunoglobulins, from
racy.
maternal bloodstream into mammary secretions during a
short period [39]. This process ceases abruptly immediately Several proteome studies have characterized the compo-
before parturition [40]. There are several factors that affect nents of colostrum in different species such as bovine and
colostrogenesis, such as species, breed, age, nutrition, litter human [12, 14, 48]. Nevertheless, little is known about co-
size, length of dry period and health status [41-43]. Moreo- lostrum and the process that takes place in the newborn,
ver, colostrum components are secreted by different mecha- namely degradation, proteolytic protection, internalization
nisms [44], so there are proteins directly produced in the and retention in the intestinal tissue. Moreover, it is neces-
mammary gland or transferred from the bloodstream (or sary to study how this primary food source passes through
both). For example, while IgG is transported to the mam- the gastrointestinal tract of the suckling neonate and how the
mary gland from the bloodstream, part of the IgA is synthe- above-mentioned factors affect the different colostrum com-
sized within the mammary gland by plasma cells which had ponents.
migrated into the gland [27], as shown in (Fig. 1).
4. MANAGEMENT EFFECTS ON COLOSTRUM AND
Proteomics is a powerful tool that can simultaneously NEWBORN RUMINANTS
analize several hundred proteins in complex mixtures; how-
ever, because of the wide range of protein concentrations and As described by Castro et al. [3] the management during
subcellular locations, there is no general protocol for the the last weeks of pregnancy is extraordinarily important,
separation of the complete colostrum proteome. Accord- because it is in this period that colostrum is secreted. There
66 Current Protein and Peptide Science, 2014, Vol. 15, No. 1 Hernández-Castellano et al.

are other factors such as litter size and number of lactation sary to separate different fractions. Colostrum proteins are
that affect final colostrum composition. Moreover, colostrum divided into three main groups according to the fraction
management in the first hours of life is crucial for the sur- where they are found: caseins, whey proteins and proteins
vival of the newborn ruminant. However, in spite of the im- from the MFGM, as summarized in (Fig. 2).
portance of these factors in the final composition and on co-
lostrum protein absorption by newborn ruminants, no pro-
teomics studies have been published on these effects.

4.1. Nutrition During Gestation

The relation between maternal nutrition during late preg-
nancy and mammary gland development, colostrum compo-
sition and volume produced after partum has been described
[49]. In fact, dairy animals that receive a balanced feed pro-
duce more colostrum than underfed animals. Regarding im-
mune components present in colostrum, nutrition manage-
ment may vary the final concentration of some immune
components. Accordingly, Awadeh et al. [50] described how
cows supplemented during late pregnancy with selenium
increased their IgG concentration in colostrum. On contrast,
Swanson et al. [51] did not observe any effect in sheep sup- Fig. (2). Schematic procedure for the separation of the three main
plemented during the late pregnancy neither in the colostrum groups of colostrum proteins. Two fractions can be obtained upon
yield nor in IgG concentration. centrifugation: Milk Fat Globule Membrane (MFGM) and skimmed
colostrum. The skimmed colostrum can be treated with ultracentri-
4.2. Litter Size and Number of Lactations fugation or acidification (or both) to obtain the last fractions: Ca-
Litter size is a very controversial effect. Csapó et al. [52] seins and Whey proteins.
found that colostrum IgG concentration was higher in twin
Hungarian White goats and Hungarian Merino sheep than in These three fractions can be isolated in two steps. The
singles. However, Argüello et al. [53] did not find differ- first step is based in the centrifugation of the colostrum in
ences in colostrum IgG concentration between Majorera order to separate the fat layer (MFGM) and the skimmed
goats with different litter sizes. colostrum that includes caseins and whey proteins. Several
centrifugation protocols may be found in the literature as
There has been some controversy on the issue of the summarized in Table 1, although some of them have only
number of lactations. While it has been shown to increase been tested in milk samples.
IgG concentration in bovine [54] and caprine colostrum [55],
Dos Santos et al. [56] and Argüello et al. [53] did not ob- After this process, the resulting skimmed colostrum
serve differences in the colostrum IgG concentration due to needs to be divided in the two last fractions (whey proteins
the number of lactations in Saanen and Majorera goats, re- and caseins). In the literature, several methods may be found
spectively . to isolate caseins from the skimmed colostrum or milk sam-
ple; however, all of them have in common the use of ultra-
4.3. First Colostrum Intake by Newborn Ruminants centrifugation, acid precipitation or the combination of both
methods. Some of the most used techniques are shown in
As described in section 2, newborn ruminants can absorb Table 2.
large molecules. Nevertheless and considering colostrum,
this special ability decreases along the first 48 hours after 5.2. High Abundant Proteins
birth. It is important to notice that the first colostrum inges-
tion is very important to acquire a correct PIT, although As described above, one of the three groups of proteins
other aspects such as colostrum IgG concentration or volume that can be found in colostrum and milk, are located in a
of ingested colostrum also take high relevance in PIT. As complex membrane, called milk fat globule membrane
reported by Chigerwe et al. [57] Holstein calves fed with 3 (MFGM) that surrounds triacylglycerols droplets
litres of colostrum between the first 4 hours after birth ac- [61].Traditionally, most of the research on MFGM has been
quire an adequate PIT. However, the same author observed focused in dairy cattle [71-75], however the interest in dif-
that calves that took their first colostrum intake between 4 ferent aspects of non-bovine milk is fast growing. For this
and 12 hours after birth also had an adequate PIT, although reason, some recent studies on MFGM of other species, such
the latter animals were fed with a high quality colostrum as goats [76], sheep [77, 78] or water buffaloes [79] may be
(IgG concentration > 100mg/mL). found in the literature.
As described by Mather [72], there are 7 HAP in the
5. COLOSTRUM FRACTIONS AND HIGH ABUN-
MFGM. Two of the most important proteins related to im-
DANT PROTEINS
munity are mucin-1 (MUC-1) and xanthine dehydro-
5.1. Colostrum Fractions genase/oxidase (XDH/XO).
As described in Section 3, there is no adequate protocol MUC-1 is a glycoprotein that is present in the apical sur-
for the separation of whole colostrum. It is therefore neces- face of epithelial cells from different organs, such as lungs,
The Colostrum Proteome, Ruminant Nutrition and Immunity Current Protein and Peptide Science, 2014, Vol. 15, No. 1 67

Table 1. Centrifugation procedures for isolation of MFGM from milk and colostrum.

Centrifugation
Specie Reference
Speed (g) Time (min.) Temp. (ºC)

Cow 5,000 15 4 [58]

Cow 15,000 15 10 [59]

Cow 10,000 15 4 [12]

Human* 1,500 20 25 [60]

Human 4,000 30 4 [61]

Ewe* 1,500 20 25 [62]

*With addition of 5g of Sucrose/100mL of milk

Table 2. Procedures to isolate caseins and whey proteins from milk and colostrum.

Centrifugation
Species Acid Precipitation Reference
Speed (g) Time (min.) Temp. (ºC)

Cow 16,500 30 4 C2H4O 2 (1N) [63]

Cow C2H4O 2 (30% v/v, pH 4.6) [58, 64]

Cow 15,000 15 10 HCl (1M, pH 4.6) [59]

Cow* 44,000 30 4 [65]

Cow 100,000 60 4 [66]

Human* 13,000 30 4 HCl (1M, pH 4.6) + 60 mmol CaCl2 [67]

Human* 189,000 60 4 HCl (1M, pH 4.3) + 60 mmol CaCl2 [68]

Cow 1,500 10 5 C2H4O 2 (1N) + CH3COONa (1N) [69, 70]

Cow 100,000 60 4 [69]

Human 10,000 15 20 HCl (1M, pH 4.6) [61]

*Centrifugation repeated twice

stomach, intestine and others [80]. This protein has been function of this protein is not completely defined, but it has
isolated in cattle [81] where its concentration is lower (40 been suggested that it may play structural and functional
mg/L) by comparison to human MFGM (729-805 mg/L) roles in the formation of the MFGM [88] and also as having
[82]. This protein was isolated in goats [83], albeit no infor- antibacterial properties in colostrum and milk, providing a
mation seems to be available on its concentration levels. source of H2O2 for lactoperoxidase [89]. Finally, this protein
Nevertheless, it has been demonstrated that it has cross- could act as an immunomodulator either causing tissue dam-
reactivity with cow MUC-1 and no cross-reactivity with hu- age and exacerbation of the inflammatory response or induc-
man MUC-1. Regarding its biological function, this protein ing expression of genes encoding, for example, adhesive
may play an important role in the protection of exposed sur- proteins, cell receptors, and components of the immune sys-
faces from physical damage and invasive pathogenic micro- tem [72].
organisms [81, 82, 84]. In addition, MUC-1 may participate Another group of proteins that can be found in colostrum
in the immune reaction in the suckling neonate because of its are whey proteins. Setting bovine milk as reference, whey
capacity of binding and sequestering pathogenic microorgan- contains more than 200 different proteins, with  -lacto-
isms within the gut lumen [82, 84]. globulin,
-lactalbumin, bovine serum albumin (BSA), im-
Concerning XDH/XO, it is essentially synthesized in the munoglobulins and lactoferrin being the major constituents
mammary gland [85, 86] and it reaches maximum values [11, 90-92]. From this group of proteins, immunoglobulins
during late pregnancy and immediately after parturition [87]. play the most important role in the innate immune transfer,
This protein has been isolated from MFGM in cows, goats although there are other proteins with important immune func-
and sheep, although differences between them have so far tions, particularly
–lactalbumin and lactoferrin [91, 93].
not been analized in colostrum. However, using milk sam- Conventionally,
–lactalbumin has been associated to
ples, it has been described that XDH/XO from sheep and the regulation of lactose production [94], Nevertheless, it has
cow MFGM have similar concentrations [74, 75, 78]. The been recently described that this protein plays also a role as
68 Current Protein and Peptide Science, 2014, Vol. 15, No. 1 Hernández-Castellano et al.

an immunomodulator in human and bovine colostrum and using less amount of sample and producing more reproduci-
milk [91, 95, 96]. The concentration of this protein in colos- ble results. On the other hand, the main disadvantage of
trum does not differ significantly between goats, cows and these techniques is the expensive cost of the equipment, in-
sheep (2.77 mg/mL, 2 mg/mL and 2.3 mg/mL, respectively) struments and reagents, not frequently available in most
[97, 98], showing that the concentration of this protein is laboratories.
approximately 1.5 times higher than those found in milk.
The other important HAP in the immune response against 6.1. Most Common Techniques Used to Identify Low
infections is lactoferrin. Konuspayeva et al. [99] studied the Abundant Proteins
differences of this protein concentration in colostrum from Approaches such as immunoabsorption [108, 109, 112],
several mammals such as camels (5.1 mg/mL), cows (0.84 isoelectric focusing (IEF) [113], affinity tagging [114] , and
mg/mL), goats (3.09 mg/mL), sheep (1.56 mg/mL) or water semi-coupled anion- and cation-exchange chromatography
buffaloes (2.1 mg/mL). Similar results were found by Nissen [59] have been used as a tool to remove HAP and increase
et al. [13] in bovine colostrum, using 2DE-LC-MS/MS and the relative abundant of LAP in proteomic analysis of com-
quantitative ELISA. The main function of lactoferrin is the plex samples such as colostrum. Despite of the removal of
binding and transport of iron ions. Additionally, lactoferrin caseins by procedures such as those described in Table 2, the
has a wide variety of biological functions, many of which do large amount of immunoglobulins and high abundant pro-
not appear to be connected with its iron binding ability [100]. teins in, for example, ruminant whey is particularly difficult
Lactoferrin works as an innate immune factor synthesized by for LAP proteomic analysis [108].
the mammary epithelium [101, 102], that provides antimi-
crobial activity (bactericide and fungicide) to the mammary As a consequence of their high abundant in colostrum,
gland and also to the newborn [103]. Due to the increase in similar isoelectric point and molecular weight, immunoglobu-
its concentration during most inflammatory reactions and lins are located in the same region of the 2DE-gel where most
some viral infections, it has been classified as an acute-phase of the LAP, such as transferrin, plasmin, lipoprotein lipase,
protein [104]. alkaline phosphatase, and others yet to be identified are also
found [59]. For this reason, it is necessary to remove HAP
With reference to casein, there are four main types (
s1,
(namely immunoglobulins) from colostrum as they can mask

s2,  and ), that are responsible for important biological
low abundant proteins. Presently, several commercial kits are
functions such as ion carriers (Calcium, Phosphate, Iron,
available to remove immunoglobulins. Golinalli et al. [63]
Zinc, Cupper), bioactive peptide precursors and immuno-
used and compared the removal efficacy of whey HAP pro-
modulators [91]. It was also demonstrated that the casein teins from colostrum using the albumin and IgG removal kit
proteolytic fragments have an antimicrobial activity [105], (GE Healthcare Life Sciences, UK) and the Vivaspin 500
suggesting that proteases may also play a role in the host ultrafiltration cartridge (GE Healthcare Life Sciences, UK)
defence. In addition, peptides derived from caseins are re- with a membrane of a molecular-mass cut-off of 100 kDa.
ceiving much attention as possible sources of natural bioac- These authors observed that the Albumin and IgG removal kit
tivity with health benefits for humans [106], probably be- was ineffective in removing IgG from the bovine colostrum
cause they stimulate the innate immune system within the whey. It is known that the albumin and IgG removal kit con-
mammary gland and prevent udder infections during the dry tains agarose-immobilized anti-IgG against human proteins.
phase [107]. The failure of these antibodies to capture the IgG present in
bovine colostrum whey could be ascribed to a lack of cross-
6. LOW ABUNDANT PROTEINS FROM COLOSTRUM reactivity of the antibodies to the bovine proteins. However,
these authors described that the treatment of colostrum whey
In addition to the high importance of HAP in colostrum,
with Vivaspin 500 removed most of the IgG and IgA, render-
there is a growing interest in LAP in order to increase the
ing possible the detection of LAP.
general knowledge about their biological role in animal and
human health [12, 63, 108]. In humans for example, a total Another common depletion technique used to deplete
of 151 proteins were recently identified, albeit the fact that high abundant proteins is the ProteoMiner kit (Bio-Rad,
83 of them had never been previously described in human USA) that consists of a unique library of hexapeptides,
colostrum or milk [109]. Probably, many of this newly iden- which bind proteins of different abundance with same capac-
tified proteins could be involved in important colostrum ity [115]. Consequently, when a complex protein mixture is
roles such as regulation of growth, nutrient transport and exposed to a ligand library, each bead with affinity to an
what it is more important, PIT. In ruminants, knowledge on abundant protein will rapidly become saturated and therefore
the identity and function of the LAP constituents of whey the vast majority of the same protein will remain unbound. In
and MFGM is still incomplete, although many of these pro- contrast, low abundant proteins will not saturate the corre-
teins may have uncharacterized biological properties [12, 59, sponding partner beads, so all of them are captured, produc-
110]. ing a progressively increase in the amount of those proteins.
After elution of the captured proteins, all proteins will be
Biochemical techniques, namely 2DE gel methods have present, but in a reduced dynamic range dependent on each
been one of the most common techniques used to identify single protein concentration before depletion [116, 117]. In
LAP in both milk and colostrum [108, 111]. However, these addition, ProteoMiner kit is a very extended method that was
techniques are starting to be replaced by other such as created for plasma and serum samples, but that has success-
iTRAQ or label-free methodologies that confer several ad- fully been tested in other samples types including urine
vantages in comparison to 2DE gel methods. These new [118], bile [119], platelets, red blood cell extract and egg
techniques are able to identify a higher number of proteins white extract [120].
The Colostrum Proteome, Ruminant Nutrition and Immunity Current Protein and Peptide Science, 2014, Vol. 15, No. 1 69

With reference to colostrum and milk, Liao et al. [67] teins and different isoforms, varying in concentration accord-
treated human colostrum and milk samples with the Pro- ing to species [127]. SAA represents one of the most con-
teoMiner kit, showing that the abundance of LAP was in- served proteins among mammals supporting the premise that
creased while some of the high abundant proteins were de- it has a basic and essential role in the innate immune system,
creased. However, these authors described that the Pro- taking part of the acute phase of inflammation [128, 129].
teoMiner kit beads had little effect on remaining casein resi- This protein has been also identified in colostrum from sev-
dues in the human colostrum and milk whey. Similarly, the eral species, namely human [130] horse, cattle and sheep
ProteoMiner kit was used to study the whey proteome of [66, 131]. It has been described that the circulating concen-
cow’s milk, obtaining an increase of the low abundant pro- tration of SAA is increased by 1000-fold within 24 to 48 h
teins [117]. It should be noticed that although this kit is able following infection/inflammation from a basal level of 5–8
to produce an enrichment of LAP, whey HAP proteins (e.g., mg/mL [132], showing the importance of this protein in in-
lactoferrin and -lactalbumin) and caseins, are still detected. flammatory processes. Moreover, a variant of this acute
For these reasons, and according to the authors, the Pro- phase protein, serum amyloid A3 (SAA-3), was found to be
teoMiner kit is a valid approach to increase the detection of expressed in mammary cells in response to pathogens or
low abundant proteins. pathogen-derived lipoteichoic acid and to be present in milk
during mastitis [131] and colostrum [131, 133] suggesting a
However, despite of the demonstrated effectiveness of the
role for this protein in host defence. Accordingly, McDonald
ProteoMiner kit in increasing the number of protein identifi-
et al. [131] found that SAA-3 concentration in cow colos-
cations in a complex protein mixture, it is necessary to re-
trum is much higher than in milk (267.45
g/mL and 2.63
mark its effect on quantitative proteomic studies, measuring

g/mL, respectively). These authors also described the con-
relative protein abundance levels between samples via stable
centration of this protein in sheep colostrum, being lower
isotope labelling [115]. Accordingly, Mouton-Barbosa et al. than in cow colostrum (62.83
g/mL and 267.45
g/mL,
[121] showed that high abundant proteins did not retain their respectively).
quantitative accuracy after the treatment with a hexapeptide
library (e.g., ProteoMiner kit), probably because they satu- In general, the SAA protein has numerous pro-
rated their peptide binding partners, resulting in a loss of the inflammatory actions: chemoattractant to neutrophils, mono-
unbound portion of the protein. On the other hand, low abun- cytes, and T lymphocytes, causing leukocyte infiltration and
dant proteins retained their quantitative accuracy after a promoting neutrophil adhesion to endothelial cells [134-136]
hexapeptide library treatment, since they did not saturate their and it stimulates neutrophils and monocytes to release not
peptide binding partners and were fully recovered. only cytokines [137, 138], but also matrix metalloproteinases
[139]. According to He et al. [140], these findings suggest a
As a result, and taking the information described before, key function for SAA not only in the establishment, but also
Proteominer kit seems to be an adequate tool to discover the in the maintenance of inflammation.
deep proteome of several samples compound by a potent and
a high mixture of proteins. Nevertheless, some considera- Fibrinogen is the precursor of fibrin, the most abundant
tions must be taken if this kit is going to be used in quantita- component in blood clots. However, this protein has also a
tive studies. defensive function, as it has been demonstrated that fibrino-
gen concentration increases during acute-phase reactions
6.2. Function of Selected Low Abundant Proteins and [141, 142]. Moreover, Yamada et al. [108] studied differ-
their Relation to the Immune System ences in low abundant proteins between cow colostrum and
milk, showing that some of them were only present in colos-
It is very important to understand how LAP from colos- trum, such as fibrinogen. Additionally, several authors have
trum can improve or modify the immune response either at described that fibrinogen can bind integrins, that are nor-
the level of the mother or at the level of the newborn, for this mally expressed by cells of the immune system, such as
reason, the function of some important LAP will be de- CD11b/CD18 [143, 144]. The CD11b/CD18 integrin recep-
scribed separately. tor (M2, Mac-1, complement receptor 3) is a member of
One of these LAP in colostrum is plasmin. This protein is the 2 integrin family, which is expressed on monocytes and
a fribrinolytic enzyme that plays an important role in the macrophages. When fibrinogen binds to CD11b/CD18, in-
dissolution of fibrin blood clots in order to prevent thrombo- tegrin causes an extensive array of cell signalling responses,
sis [122]. Nevertheless, this protein has been identified not namely the activation of the nuclear factor kappa-light-chain-
only in blood, but also in colostrum and milk [66, 123]. As enhancer of activated B cells and mitogen-activated protein
described by Dupont et al. [123], bovine colostrum contents kinase/phosphatidylinositol 3-kinase, that mediate adhesion,
10 times more plasmin (0.49
g/mL) than milk (0.04 migration, chemotaxis and phagocytosis [145]. All these

g/mL). In bovine, sheep and goat milk, plasmin is essen- reasons, confirm that fibrinogen and fibrin from colostrum
tially identical to those found in blood [124]. Additionally, may result beneficial to the newborn immune system effi-
other authors have observed that plasmin has immune activ- ciency.
ity, as it contributes to the neutrophil migration to the site of Trypsin inhibitor (TI), also known as 1-antitrypsin, is a
infection [125]. In agreement with these findings, an increase protein that reduces biologically active trypsin [146], de-
in plasmin concentration in blood and milk during acute creasing proteolysis. This protein has been described in bo-
mastitis in lactating dairy ewes has been described [126]. vine colostrum using different proteomics techniques, such
Another interesting protein is serum amyloid A (SAA). as 2DE gel electrophoresis [108] or LC-MS/MS [13]. It has
This protein is normally found in complexes with lipopro- been studied that colostrum contains 100 times more TI than
70 Current Protein and Peptide Science, 2014, Vol. 15, No. 1 Hernández-Castellano et al.

milk [147, 148], probably to protect against the proteolytic trum and their relation to PIT and the immune system devel-
cleavage, allowing the absorption of the intact immune opment. Moreover, further studies will be necessary in order
components by the newborn ruminant. to understand the biological process of colostrum proteins
uptake trough the gut in newborn ruminants, particularly in
The lipopolysaccharide-binding protein (LBP) is one of
less studied domestic ruminants such as sheep, goats and
the most produced during infections with Gram-negative
water buffalo.
bacteria [149]. Additionally, LPB seems to actively partici-
pate during gram-positive bacterial infections [150]. This
protein has been detected in cow colostrum using 2D-LC- CONFLICT OF INTEREST
MS/MS [13] and then quantified using ELISA (0.85 mg/mL, The authors have no conflict of interests regarding com-
approximately) by the same authors. No references about the mercial products, or any other aspects, referred to in this
presence of this protein has been found neither goat nor
manuscript.
sheep colostrum. The main function of this protein is to bind
bacterial lipopolysaccharides (LPS) expressed on the outer
cell wall of bacteria, acting as a carrier for LPS and to help ACKNOWLEDGEMENTS
control LPS-dependent monocyte responses [14]. Author Hernández-Castellano, L.E. acknowledges finan-
Antimicrobial peptides (AMPs) are defined as such for cial support from the Formación del Profesorado Universi-
their capacity to rapidly inactivate infectious agents [151]. tario (FPU) program (Ministry of Education, Madrid, Spain).
Most AMPs act by disrupting the integrity of the microbial Author Almeida, A.M. acknowledges funding from Funda-
membranes [151]. The two major AMP families in mammals ção para a Ciência e a Tecnologia in the form of grant
are the defensins [10] and another group of cationic mole- SFRH/BPD/90916/2012 and Project PTDC/CVT/116499/
cules, classified as cathelicidin peptides [152, 153]. 2010 - Lactation and milk production in Goat (Capra hir-
Defensins are a group of small antimicrobial peptides cus): identifying molecular markers underlying adaptation to
that interact with the adaptive immune response [154]. These seasonal weight loss.
peptides are produced constitutively and/or in response to
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Received: October 13, 2013 Revised: October 13, 2013 Accepted: November 19, 2013