This is an open access article published under an ACS AuthorChoice License, which permits

copying and redistribution of the article or any adaptations for non-commercial purposes.

Review

Cite This: Chem. Rev. 2018, 118, 7867−7885 pubs.acs.org/CR

Capillary Electrophoresis Separations of Glycans

Grace Lu, Cassandra L. Crihfield, Srikanth Gattu, Lindsay M. Veltri, and Lisa A. Holland*
C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States

ABSTRACT: Capillary electrophoresis has emerged as a powerful approach for

carbohydrate analyses since 2014. The method provides high resolution capable of
separating carbohydrates by charge-to-size ratio. Principle applications are heavily focused on
N-glycans, which are highly relevant to biological therapeutics and biomarker research.
Advances in techniques used for N-glycan structural identification include migration time
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

indexing and exoglycosidase and lectin profiling, as well as mass spectrometry. Capillary
electrophoresis methods have been developed that are capable of separating glycans with the
Downloaded via SHANGHAI JIAO TONG UNIV on March 28, 2024 at 04:22:12 (UTC).

same monosaccharide sequence but different positional isomers, as well as determining

whether monosaccharides composing a glycan are alpha or beta linked. Significant
applications of capillary electrophoresis to the analyses of N-glycans in biomarker discovery
and biological therapeutics are emphasized with a brief discussion included on carbohydrate
analyses of glycosaminoglycans and mono-, di-, and oligosaccharides relevant to food and
plant products. Innovative, emerging techniques in the field are highlighted and the future
direction of the technology is projected based on the significant contributions of capillary electrophoresis to glycoscience from
2014 to the present as discussed in this review.

CONTENTS 6.2. Realizing the Full Power of Electrophoresis

and MS 7880
1. Introduction 7867 6.3. Future Directions 7881
2. Background 7868 Author Information 7881
2.1. Carbohydrate Fundamentals 7868 Corresponding Author 7881
2.2. Capillary Electrophoresis Fundamentals 7869 ORCID 7881
2.3. Performance of Capillary Electrophoresis Notes 7881
Relative to Other Methods 7870 Biographies 7881
3. Carbohydrate Derivatization 7870 Acknowledgments 7881
3.1. Advances in Labeling 7870 References 7881
3.2. Analytical Technology To Improve Sample
Processing 7871
4. Methods To Identify Glycans 7871
4.1. Calibration of Migration Time with Ladders 1. INTRODUCTION
and Standards 7871
Carbohydrates are fundamental to signaling, structure, and
4.2. Identification with Exoglycosidases and
energy. These molecules play important roles in renewable
Lectins 7873
energy,1 disease,2−4 aging,5 food,6 and therapeutics.7 The
4.2.1. Identification with Exoglycosidases 7873
critical nature of glycans in signaling8 supports the use for
4.2.2. Glycan Identification with the Use of
disease diagnosis, prognosis, and therapeutic intervention.
Lectins 7874
Glycoscience encompasses a diverse range of applications
4.3. Structural Verification with MS 7874
such as mapping glycopeptides in protein targets or elucidating
5. Applications 7876
the role of glycosylation in signaling and ligand−receptor
5.1. Biomarkers 7876
binding. Insight into the processes that lead to changes in
5.1.1. Changes within and across Individuals 7876
glycosylation continues to become increasingly important in
5.1.2. Autoimmune Disorders 7876
medical research9 and biopharmaceutical manufacturing.10
5.1.3. Cancer 7877
Glycosylation modulates biological activity11 and strongly
5.1.4. Biomarker Studies of Cells 7878
affects the antibody effector function. This holds the potential
5.2. Application to Biological Therapeutics 7878
to dramatically improve drug performance7 but requires
5.3. Other Carbohydrates 7878
enabling technologies, including capillary electrophoresis
5.3.1. Glycosaminoglycans 7878
5.3.2. Food and Plant Carbohydrate Analyses 7879
6. Emerging Techniques and Future Directions 7879 Special Issue: Carbohydrate Chemistry
6.1. High Throughput Structural Characterization Received: November 7, 2017
of Biological Therapeutics 7879 Published: March 12, 2018

© 2018 American Chemical Society 7867 DOI: 10.1021/acs.chemrev.7b00669

Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

(CE),12,13 to monitor microheterogeneity throughout manu- 2. BACKGROUND

facturing.13
2.1. Carbohydrate Fundamentals
The interest in CE for glycoscience applications is evident by
the recent high activity in published reviews in 2016 and 2017 It is easier to appreciate the analytical challenges associated
alone.14−18 This review focuses on the analysis of released with carbohydrate analyses by considering the complexity of
carbohydrate structures. A brief description is presented in this
oligosaccharides as a means to highlight the dynamic and
review, which centers on factors important to CE. Other
rapidly evolving advances in CE instrumentation and method-
sources may provide additional information regarding a detailed
ology. In 2015, several industrial and academic laboratories description of glycan nomenclature and structure,33 as well as
participated in an interlaboratory test, which involved an N- an overview of different nomenclature and fragmentation
glycan mapping exercise with a protein test sample and patterns observed in MS.34
demonstrated good reproducibility in area and migration Monosaccharides, which are the building blocks for
time.19 Currently, commercial kits have made use of the polysaccharides such as glycans and glycoforms, can be roughly
technology more routine in industry by streamlining sample classified to contain an empirical formula of (Cx(H2O)n). A vast
preparation for higher throughput.20−24 These advances are array of different monosaccharides exist,35 but for the purposes
serendipitous as a healthy rate of growth is expected for the of this review, only 6 structures are presented in Figure 1 to
antibody pharmaceutical market, which is estimated to reach an
annual global value of $125 billion USD in 2020.25 This creates
an even more pressing need for high throughput analyses,
which can be met by CE.
There are several advantages of CE. High electric field results
in short separation times and high efficiency, in the absence of
Joule heating.26 Injected sample volumes are in the femtoliter
to nanoliter range. Only a few milliliters of running buffer are
required for a 24-h period. Separations performed on
commercial instruments are automated. Detection limits with
laser-induced fluorescence are in the femtomolar range. Typical
instruments contain a single capillary with temperature control
of the capillary and solutions. Multiple capillaries can be used in
a single instrument to increase the throughput. For example,
instruments used for DNA sequencing can hold from 16 to 96
capillaries.27−31 Injections can even be multiplexed to deliver
packets of sample within a single run to eliminate dwell times.32
Innovations in instrumentation and in applications of the
methodology continue and can be observed as a push to make
the technique more accessible to researchers through Figure 1. Six representative structures of D conformer monosacchar-
commercially available solutions to interfacing CE with a ides. Glucose, mannose, and galactose are common unsubstituted
mass spectrometer, as well as databases and software that hexose saccharides. Fucose has one less hydroxyl group and is termed
increase the ease of data analysis. Analytical techniques are a deoxy-hexose sugar. N-Acetylglucosamine and N-acetylneuraminic
continually adapted to meet the growing challenges in acid represent substituted hexose saccharides.
carbohydrate analysis and CE plays a unique role in
glycoscience research. demonstrate the subtle differences in these structures. The
This review is an exhaustive update of the most recent position of the hydroxyl substituents is the only difference
activity in CE glycoscience applications, covering advances from among hexose saccharides: mannose, glucose, and galactose.
2014 through 2017. Recognizing that the field is multi- Examples of substituted hexose saccharides include N-
disciplinary, attention is given to fundamental principles of acetylneuraminic acid and N-acetyl glucosamine. Fucose is an
glycan structure as well as CE separations. Advances in labeling example of deoxy-hexose saccharide.
and the development of highly automated, high throughput Polymerizing these saccharide monomers dramatically
methods of sample preparation are noted. Substantial increases the structural diversity, as well as the complexity of
consideration is given to the three primary approaches to carbohydrates. Oligosaccharides and polysaccharides can vary
identifying glycan peaks, including migration time indexing, by the monomer structure. The linkage orientation can be
alpha or beta, and the position of the linkage can differ, as
enzyme or lectin profiling, and mass spectrometry (MS). A
shown in the trisaccharides depicted in Figure 2. Although the
discussion of advances in labeling and in particular in creating known biosynthetic pathways of classes of oligosaccharides
highly automated high throughput methods of sample limit the variety of structures that are physiologically relevant, a
preparation is included. A focus is placed on cutting edge range of diverse carbohydrate structures occur, which is why
applications and technologies. A review of applications for these molecules play a substantial part in signaling. A
biomarker discovery, characterization of therapeutics, and consequence of this diversity is that isomeric structures are
applications to food and pathogen analyses is given. The not only possible but highly probable. Amidst this complexity
report concludes with areas of emerging CE technologies that in carbohydrate structure, other repeating structural motifs have
will have a long and sustaining effect in the field of been classified. This includes linear polysaccharides composed
glycosciences with further development. of repeating disaccharide units that are glycosaminoglycans
7868 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

by targeting the reducing end of polysaccharide structures, as

shown in Figure 3. This ensures the molecules are labeled with
a single fluorophore using standard reductive amination. The
addition of a charged tag on the N-glycan structure is
serendipitously compatible with CE.
2.2. Capillary Electrophoresis Fundamentals
Capillary zone electrophoresis separations are well-suited to
biomolecular separations because the method is based on
transport in an electric field.36 Electroosmotic flow (EOF) and
electrophoretic mobility are the two transport mechanisms that
are fundamental to CE. EOF is the bulk flow of the background
electrolyte. In a bare fused silica capillary EOF moves from the
anode toward the cathode due to the negative surface charge of
the capillary and the presence of the electric field. Electro-
phoretic transport is a function of charge-to-size ratio. Analyte
is attracted to either the anode or cathode and the size of the
Figure 2. Two structures of sialyllactose that differ only by the linkage molecule determines the speed of transport. These two modes
between the sialic acid and galactose monosaccharides. The structure of transport are superimposed in most CE separations. In a CE
in panel A is 6′-sialyllactose, which is composed of an α2−6 linkage. separation with the anode at the site of injection in a bare fused
The structure in panel B is 3′-sialyllactose, which is composed of an silica capillary, when EOF is present the order of analyte
α2−3 linkage. migration is small positively charged molecules, larger positively
charged molecules, comigrating neutral molecules, large
(mucopolysaccharides). This class of polysaccharides, which negatively charged molecules, and smaller negatively charged
plays various roles in life including motor function, cell growth, molecules.
and anticoagulation, is composed of many familiar molecular Separations of carbohydrates by CE are unique because a
classes such as heparin/heparan sulfate, chondroitin/dermatan majority of saccharides are uncharged, except glycans
sulfate, hyaluronic acid, and keratin sulfate. containing acidic sugars (e.g., N-acetylneuraminic acid,
A predominant class of carbohydrates in biomarker research glucuronic acid, or iduronic acid). Additionally, carbohydrate
and biological therapeutics are asparagine-linked carbohydrates molecules lack a chromophore and cannot be detected with
or N-glycans. Serine and threonine glycans (O-glycans) also absorbance detection in the UV range. These confounding
exist, but their analyses by electrophoresis has not accelerated factors are addressed by labeling the carbohydrates with a
to the same degree as seen for N-glycans owing to difficulties fluorophore. The fact that most fluorophores are charged is an
associated with glycan labeling via reductive amination advantage in CE because it ensures that the labeled
following chemical release of the glycan by β-elimination. carbohydrate migrates in the electric field. The fluorescent
The common feature of N-linked glycans is a core structure label 8-aminopyrene-1,3,6-trisulfonate (APTS) was among the
composed of a combination of N-acetylglucosamine residues first dyes reported for oligosaccharide analyses with CE37 and
and mannose residues as shown in Figure 3.33 Complex N- has an excitation maximum near the 488 nm line of an argon
ion laser. Labeling with APTS, which is shown in Figure 4, has
been well established.38−40 Covalent modification of oligosac-
charides by reductive amination produces singly labeled
product at nearly 100% labeling efficiency.

Figure 3. Depiction of an N-glycan, which is composed of several

Figure 4. Simplified mechanism of reductive amination as it is used to
monosaccharides, removed from an antibody. The reducing end (B),
which is located where the N-acetylglucosamine is cleaved from the label carbohydrates with APTS using a reducing agent (sodium
cyanoborohydride).
antibody, is the target for the attachment of fluorophores, such as
APTS.
Once labeled glycans are negatively charged, the separation is
glycans vary by the presence of fucose, galactose, additional adjusted to rely on electrophoretic transport to accentuate
branching and bisecting N-acetyl glucosamine, sialic acid differences in charge-to-size ratio. As depicted in Figure 5,
capping, and other modifications such as polylactosamine neutral glycans harbor three additional negative charges
capping. Although N-glycans represent only a single category of following conjugation to APTS. Sialylated glycans labeled
carbohydrates, there is a strong focus on these structures in CE with APTS are even more negative and as a result have an even
because they are a post-translational modification with heavy faster migration. The EOF is suppressed by eliminating the
implications in physiological function. This makes N-glycans surface charge on the capillary wall through surface
powerful for biomarker discovery and for practical applications modification. The electric field is applied under reversed
in biological therapeutics. In spite of the complexity and polarity to drive anions toward the detection window. Using
diversity of N-glycan structures, N-glycan labeling is simplified the simplest relationship between molecular weight and
7869 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

purpose of this study was to establish the benefits of these

available technologies since focusing on particular technologies
affects the findings of large scale studies as well as the resource
investment. Several important advantages of CE were identified
by the authors. First, the high resolution of CE enabled isomer
separation and linkage analysis. Second, quantification was
achievable only with CE and the liquid chromatography
method coupled to fluorescence detection. Third, by using
instruments with 16-capillary arrays, the authors achieved
extraordinary throughput relative to both liquid chromatog-
raphy methods. The cost of the CE instrument, as reported by
the authors was on par with that of liquid chromatography and
with low-end MS approaches. The authors noted that the CE
method offered substantially reduced cost per sample.
Limitations identified by the authors included poor acceptance
by the glycomics community and the lack of a large structural
database.
Other reports have compared the performance of CE to
separation techniques used to analyze N-glycans. Reusch et al.
compared the performance of capillary gel electrophoresis with
fluorescence detection against hydrophobic interaction liquid
chromatography (HILIC) separations coupled with fluores-
Figure 5. Basic diagram of the capillary electrophoresis system cence using two different labels, demonstrating good agreement
commonly used to separate APTS-labeled glycans. Separations are among these approaches as well as the advantage of CE to
performed in reverse polarity (cathode to anode) under suppressed separate positional isomers. 27 A complementary study
electroosmotic flow using a coated capillary. APTS-labeled glycans are
compared CE with fluorescent detection to anion exchange
separated by charge-to-size ratio in the order of ascending hydro-
dynamic volume as shown in the inset and quantified using an with pulsed amperometric detection and HILIC with
electropherogram generated by laser-induced fluorescence. fluorescent detection, which served as the reference.49 Different
modes of CE were evaluated including capillary zone
electrophoresis (separations in the presence of EOF), capillary
molecular size, the electrophoretic mobility, μeph, is a function gel electrophoresis (separations under reversed polarity with
of molecular size as described in eq 1 suppressed EOF and a gel additive), and DNA fluorophore
μeph = q/6πηr (1) assisted CE (capillary gel electrophoresis at an elevated
temperature using a genetic analyzer). The authors indicated
where q is the charge, η is the viscosity of the background only minor differences in accuracy, precision, and separation
electrolyte, and r is the hydrodynamic radius of the molecule.41 performance.49
Viscosity plays a role in frictional drag (see eq 1) and is one Comparisons of separation-based methods for N-glycan
strategy to achieve the high resolution needed to distinguish analyses using significantly larger sample sets to evaluate a
oligosaccharide isomers. Additives are often included in the wider range of glycosylation present on IgG antibodies were
background electrolyte to improve resolution. The carbohy- also reported. Adamczyk et al. explored the differences in
drate separation mechanism facilitated by the use of linear gels separation based approaches of CE, reversed phase liquid
is solely based on charge-to-size ratio and is not size-based chromatography, and HILIC by comparing the results obtained
sieving, which is observed for larger biomolecules such as for therapeutic IgG Fc-glycosylation profiles from different
proteins and DNA.42 As it is applied to carbohydrate analyses, healthy mammalian species.50 The results of their work
the separation technique has been interchangeably referred to demonstrated that CE and HILIC had similar performance
as CE or capillary gel electrophoresis when a gel is included in and were both better suited to resolve complex mixtures than
the background electrolyte. Fluorophore assisted carbohydrate reversed phase liquid chromatography.50 In a separate report,
electrophoresis has also been used, although this nomenclature Mahan et al. compared capillary gel electrophoresis to HILIC
can be misleading as the method may be accomplished using demonstrating that while both methods yield comparable
slab gel43,44 as well as CE.45 results, electrophoresis was more cost-effective, consumed less
2.3. Performance of Capillary Electrophoresis Relative to sample, and was operated with higher throughput.29 The
Other Methods authors demonstrated that the method was suitable to identify
A variety of analytical tools are used significantly in differences in glycosylation profiles for Fab fragments from
glycosciences including chromatography,18,46 mass spectrome- polyclonal antibodies expressed within species and that
try,18,46−48 ion mobility,18,47 and lectin arrays,48 and CE has glycosylation profiles are dramatically different across human,
developed into a powerful approach to complement existing rhesus, and mouse.29
technology. In 2014, Huffman et al. reported a comprehensive
assessment of the use of CE relative to reversed phase 3. CARBOHYDRATE DERIVATIZATION
chromatography coupled with fluorescence detection matrix
assisted laser desorption ionization-time-of-flight MS versus 3.1. Advances in Labeling
liquid chromatography−electrospray ionization−MS. Each of Glycan labeling with APTS remains the method of choice for
these techniques was evaluated by measuring N-glycans from CE separations.19,27,29,31,40,50−73 Glycan labeling with APTS
IgG molecules in plasma samples from 1201 individuals.30 The through reductive amination is well-established and is preferred
7870 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

over other strategies, including Michael addition or hydrazide74 liberated as a positively charged glycosylamine. This glycone
or oxime formation.75 A recently reported modification of product ion pairs with the carboxylic acid beads and is easily
reductive amination chemistry is the use of catalysts to facilitate separated from the spent enzyme reaction and cleanly
the direct transfer of hydrogen.57 The use of catalysts is transferred to solution ideal for APTS labeling. During
important to automated parallel processing and subsequently purification, the reaction solution is diluted to contain a high
high throughput because it obviates the generation of hydrogen acetonitrile composition (80%). Under this condition, the
cyanide formed using sodium cyanoborohydride in the beads support hydrophilic interaction, retaining glycans due to
presence of acid. molecular crowding. An alternative strategy to purify labeled
In addition to APTS, other labeling reagents can be used glycans from the labeling process utilizes solid phase extraction
with CE separations of carbohydrates. Recently, 2-amino- materials. A fritless in-line solid phase extraction device was
benzoic acid was used with a helium cadmium laser rather than designed that coupled large diameter anion exchange packing
an argon ion laser,76 as well as for UV−visible absorbance material into the separation by sandwiching the material
detection77 or CE coupled with MS.78 The fluorescent label 7- between two narrow inner diameter capillaries.70 The in-line
amino-4-methylcoumarin79 was also reported with an UV light fritless cartridge was used to enrich anionic samples prior to the
emitting diode excitation source. This dye is neutral following CE separation and was successfully applied to APTS-labeled
conjugation and labeling, so the use of boric acid, which glycans.70 Finally, slab gel electrophoresis was reported for
complexes with diols to form a negative complex in order to glycan purification by excising gels and extracting the labeled
separate glycans, was required.80 glycans.51
Despite the expansion of glycan analysis to the use of other
dyes, APTS is still the most commonly utilized dye for glycan 4. METHODS TO IDENTIFY GLYCANS
analysis by CE for several practical reasons. The fluorescence of Several methods have been developed in an effort to identify
the APTS labeled glycans is 40-fold higher than that of the structure and linkage composition of unknown glycans in
unconjugated APTS.37 Additionally, the background interfer- samples. These techniques fall into the broad categories of
ence from biomatrices can be reduced because the excitation calibration of migration time with ladders and standards,
wavelength of APTS is in the visible range (λex = 488 nm), exoglycosidase and lectin reactions, and MS analysis. This
whereas the other dyes are excited in the ultraviolet range, 2- section details the fundamentals and recent advances of these
amino benzoic acid (λ ex = 325 nm) and 7-amino-4- techniques as they are critical to glycan identification in current
methylcoumarin (λex = 354 nm). and emerging technologies.
3.2. Analytical Technology To Improve Sample Processing 4.1. Calibration of Migration Time with Ladders and
Given the significance of antibody therapeutics and a clear need Standards
to assess glycosylation on a high throughput scale, the CE utilizes a migration time index for glycan identification.
enzymatic and chemical steps performed in research Briefly, the migration of the analyte is best referenced to a size
laboratories to deglycosylate, label, and purify samples are a ladder generated by a homologous series of a linear glucose
bottleneck to routinely implementing CE separations in polymer.82 Some aspects of the separation introduce variability
pharmaceutical processing. Serious effort has been invested in in the migration time, which can be accounted for using a size
automating these sample handling steps required to achieve the ladder as a standard. The apparent mobility of the analyte is the
labeled glycan products. Purification of antibody therapeutics sum of the electrophoretic mobility and bulk EOF. Although
from crude lysate using Protein A extraction cartridges and the EOF is suppressed, a small component is present and will
labeling are accomplished with a combination of commercial change from run to run with any changes in the surface
products or with complete kits.63 Other efforts to increase characteristics of the capillary wall. Adsorption of biomolecules
throughput include different strategies to immobilize the to the surface, a change in the ionic strength of the background
enzyme or glycan during processing. electrolyte, or variation in the pH, for example, due to
Prior to analysis, N-linked glycans are typically released from electrolysis in the anodic and cathodic reservoir, will contribute
glycoproteins enzymatically, although methods of chemical to this modest variability in EOF. Referencing the analyte to the
release have recently been described based on simple oxidative ladder solves any issues with reproducibility in the migration
release with sodium hypochlorite.81 PNGase F, the enzyme time. Glucose unit (GU) values have been used to quantify the
commonly used for the release of N-glycans, has been impact of the number of monosaccharides and the mono-
immobilized using commercially available glutathione resin saccharide linkage in CE separations and to estimate the
and a glutathione S-transferase fusion protein of PNGase F.65 unknown glycan structure (hydrodynamic size) as early as
The authors demonstrated faster reaction kinetics for the 1996,83,84 with a series of improvements being published.
immobilized enzyme and complete turnover for both The approach is identical to the use of Kovat’s index as a
immobilized and free solution PNGase F.65 These automated referencing tool in gas chromatography. Indexing migration
steps have been fully integrated with a Biomek FXP Laboratory time improves the reproducibility of separations based on the
Automation Workstation.64 same method. It also is effective for comparing migration times
Different commercial kits are available to prepare glycans across separations performed with different methods, as was
with improved workflow.20−24 Automated processing for demonstrated for different field strengths, effective separation
labeling continues to advance beyond the use of HILIC lengths, separation temperatures, surface coatings, and injection
extraction cartridges to purify labeled glycans. Magnetic beads modes.85 Traditionally, a complete glycan ladder is used to
(Agencourt Cleanseq magnetic beads from AB Sciex) have estimate the relative size of the sample using the migration time
been used to automate 5 serial steps of labeling.40 The of the standard oligomer and the analytes to address an
commercial beads are manufactured to contain carboxylic acid experimentally observed change in ladder migration associated
functional groups. During deglycosylation, the glycone is with a helical turn that occurs in maltooligosaccharide
7871 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

structures above a degree of polymerization of 7.85 However, as GU values is attributed to the difference in activation energy for
shown in Figure 6, the approach is simplified to only three co- linear and branched glycans, which is 0.4 and 1.5 J/mol/Å3,
respectively.54 The concept of exploiting differences in the
activation energy of different glycan structures by changing
separation temperature was utilized to optimize the resolution
achievable for N-glycans of biotherapeutic interest.87
Furthermore, the virtual maltooligosaccharide ladder was
developed using a triple-internal standard approach in order to
circumvent interference when co-injecting the glycan ladder
with target analytes or the need to perform a separate run for
the standard glycan ladder. These three internal standards
include maltose (DP2), maltotriose (DP3), and maltopentade-
caose (DP15). The DP3 was added to the cleaved N-glycans in
order to estimate labeling as well as the injection efficiency.
This enabled its use as an internal standard as well as a
migration standard. The APTS labeled DP2 and DP15, which
were not included in the sample reaction, bracketed the target
analyte in the electropherograms without overlapping with the
glycan peaks. The virtual glycan ladder from DP2 to DP7 was
estimated using the migration time difference between the two
Figure 6. Electropherogram of three internal standards (DP2, DP3,
injected standards, GUDP2 and GUDP3. The ratio of the
and DP15 are maltose, maltotriose, and maltopentadecaose,
respectively) and APTS labeled N-linked glycan released from migration time differences between two consecutive peaks
human immunoglobulin G in the upper trace. The representative (DP < 7) to DP2 and DP3 was reproducible (RSD < 1%). On
electropherogram of maltooligomers in the lower trace is aligned with the other hand, the GU values for oligosaccharides with more
DP2, DP3, and DP15 in the upper panel. Reprinted with permission than 7 glucoses (DP8−DP15) were evaluated using the
from ref 85. Copyright 2016 American Chemical Society. migration time of DP15 in the sample traces because
maltooligosaccharides with more than 7 glucoses form helical
injected size standards to accurately index migration time structures, and the migration time differences between two
without the drawbacks of ladder and sample overlap due to consecutive oligomers remain constant.82 Therefore, the virtual
comigration.85 Additionally, one standard also serves to ladder, as well as the injection efficiency, can be estimated using
normalize variations in the sample injection.85 the triple-internal standard.
The calculation of the GU value is straighforward.56 As High precision of the measured GU values obtained from the
shown in eq 2, the glucose index of the specified glycan analyte, triple-internal standard approach allows identification and
GUx prediction of the structures based on a direct comparison of
t − tN GU values between known standards and the unknown glycan.
GUX = GN + X The N-glycan profile of formalin-fixed paraffin-embedded
tN + 1 − tN (2) mouse tissue samples was identified using the combination of
where GN is the number of maltose in the adjacent exoglycosidase and the GU values.58 The sample included
maltooligosaccharide ladder peak, tX, tN, and tN+1 are the neutral and mono- and disialo N-glycans. The presence of
migration times of the target glycan and the N and N + 1 terminal sialic acid, carrying one negative charge, led to shorter
number of maltooligomers, respectively. The use of a glycan migration times. Therefore, GU values in the range of 4−6, 7−
ladder and GU values was further improved with the built-in 9, and 9−13 indicate disialo, monosialo, and neutral N-glycans,
database GUcal for high throughput analysis,56,85 reported as respectively. The N-glycan patterns after exoglycosidase
www.gucal.hu.86 The program automatically calculates the GU treatment were identified using GU value and a publicly
values of peaks in the electropherogram upon loading the accessible database.
ASCII file of both standard maltooligosaccharide ladder and An alternative platform to analyze N-glycan sequence using
sample traces. multiplexed capillary gel electrophoresis was developed based
GU values are also applied to evaluate the impact of on a DNA sequencing instrument. Software glyXtool30,66,88 and
temperature on hydrodynamic size of both linear and branched glyXalign30,89 were developed for N-glycan analyses in order to
N-glycans using CE.54,87 The experimental results showed that address issues with shifts in migration time and peak
GU values changed as a function of temperature from 20 to 50 assignment. GlyXalign and glyXtool identify the peaks and
°C using background electrolytes prepared with or without align the electropherograms based on the internal standard,
linear polymer additives. The temperature dependence of the which is composed of single-stranded DNA fragments ranging

Table 1. Examples of Commercial Exoglycosidases

monomer enzyme linkage selectivity

N-acetylneuraminic acid N-acetylneuraminidase: α2−3, α2−3,6, α2−3,6,8, α2−3,6,8,9
galactose galactosidase: β1−3, β1−4, β1−3,4, β1−3,6, β1−4,6
N-acetylglucosamine N-acetyl-glucosaminidase: β1−2,3,4,6
mannose mannosidase: α1−2, α1−2,3, α1−6, α1−2,3,6
fucose fucosidase: α1−2, α1−6, α1−3,4, α1−2,4,6, α1−2,3,4,6

7872 DOI: 10.1021/acs.chemrev.7b00669

Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

in size from 35 to 500 bases (GeneScan-500 LIZ Size different aspects of the analysis. The formation of sodium
Standard). As a result, high throughput analysis for N-glycan adducts or introduction of nonvolatile salts is a concern for CE
identification was achieved and applied for diagnostic separations coupled with MS. The potential for some
purposes.88 exoglycosidase enzymes to exhibit transferase activity, reattach-
4.2. Identification with Exoglycosidases and Lectins ing the liberated monomers to the product, can be alleviated
4.2.1. Identification with Exoglycosidases. Exoglycosi- with in-line reactions. Optimized reaction conditions with long
dases are enzymes that cleave an oligosaccharide residue from incubation times allow the use of lower amounts of enzyme,
the nonreducing end of a glycan, proteoform, or oligosacchar- which can be important for expensive enzymes, such as those
ide (see Figure 3). These enzymes are selective for specific with high specificity. This also accounts for differences in
oligosaccharide monomers. The enzyme may have additional reaction rates inherent with different substrates. For example,
selectivity, for example, for the linkage orientation (α vs β) or neuraminidase derived from Clostridium perf ringens (C. welchii)
the position of the carbon−carbon linkage (e.g., α2−3 vs α2−6 exhibits higher rates of cleavage dependent upon the
sialic acid as shown in Figure 2). Conversion of a carbohydrate neuraminidase linkage with α2−3 > α2−6 > α2−8. This is in
by an exoglycosidase is only possible if the exoglycosidase contrast to rates observed with neuraminidase derived from
specificity matches the characteristics of the terminal monomer. Arthrobacter ureafaciens or from Vibrio cholera, which exhibit
As summarized in Table 1, a number of exoglycosidases are cleavage rates of α2−6 > α2−3 > α2−8.
commercially available with different specificity. The conversion Exoglycosidases can also be integrated into the CE separation
of substrate to product is harnessed with CE by analyzing the to streamline carbohydrate identification, reducing the amount
carbohydrate before and after enzyme treatment. The of enzyme as well as the time required for enzymatic
formation of product is observed as a shift in migration time. conversion. This was recently utilized as a means to quantify
This is depicted conceptually in Figure 7A, where galactosidase the rate of enzymatic cleavage as the Michaelis−Menten
constant. Michaelis−Menten constants are determined by
measuring the rate of product formation at different substrate
concentrations. A plot of the reaction velocity against substrate
concentration generates a hyperbolic curve. The substrate
concentration that produces the half-maximal reaction velocity
is the Michaelis−Menten constant. Neuraminidase enzymes
with different sialic acid linkage specificity were evaluated for
Michaelis−Menten constants for α2−3 versus α2−6 sialyllac-
tose.77 As measurement requires that the substrate concen-
tration is on the order of the Michaelis−Menten constant,
analyses of neuraminidase required the carbohydrates to be
labeled with a UV−visible active tag (2-amino benzoic acid).
This facilitated the use of substrate concentrations ranging from
0.4 to 7.4 mM, producing quantifiable levels of the lactose
product.
With knowledge of the Michaelis−Menten constant, the
method of in-line enzymatic sequencing was used to evaluate
complex N-glycans. These studies were accomplished with
fluorescent detection of APTS-labeled N-glycans, which made
the method applicable to nanomolar N-glycan samples. Unique
Figure 7. Conceptual diagrams of online enzyme and lectin reactions to this study was the use of a thermally reversible nanogel to
in-capillary. Panel A depicts a galactosylated triantennary glycan pattern the enzyme in specific locations in the separation
separated in the absence and presence of galactosidase. Upon cleavage capillary either in aqueous background electrolyte or in capillary
of the terminal galactose residues, the glycan migrates faster due to
mass loss. Panel B depicts galactosylated triantennary glycan in the
filled entirely with nanogel. Substrate was driven through the
absence and presence of Erythrina cristagalli lectin. Upon binding to nanogel, and incubation was controlled electrophoretically by
the terminal galactose residues, the glycan is not detected. repeatedly applying voltage in forward and reverse polarity in
order to cycle the analyte through the region of the capillary
that contained enzyme. The study demonstrated a means to
enzyme cleaves terminal galactose residues from an N-glycan. It extend the lifetime of enzyme preparations at low concentration
is possible to elucidate the sequence and linkage of from a few hours to over 1-month.77 Exoglycosidase with high
oligosaccharides following treatment with appropriate exogly- specificity for α2−3 sialic acid linkages was used to evaluate the
cosidases. These analyses are performed off-line or online with relative composition of sialic acid linkage. A significant finding
the CE. of this work is that the enzyme considered general for α2−3
The benefit of modifying N-glycan samples prior to CE and α2−6 sialic acid could also be used to quantify the relative
separations using enzymes was recognized early.90 Applications composition of sialic acid linkage by harnessing the lower
with CE have been performed off-line with a variety of reaction rate observed for α2−6 linkages as compared to α2−3
enzymes28,59,88,91 and typically focus on removal of sialic linkages. This resulted in substantial cost savings as enzymes
acids52,66,92 in some cases as a means to reduce complexity of with linkage specificity are more expensive than enzymes that
the CE separation.92 These reactions are typically performed cleave a particular saccharide residue regardless of linkage. The
overnight at an elevated temperature and a specified pH. Other use of nanogel to pattern enzyme was extended to galactosidase
considerations for off-line reactions include vendor added in order to distinguish terminal galactose with a β1−3 versus
stabilizers or other additives with the potential to interfere with β1−4 linkage in complex N-glycans.93 Again, mixing during the
7873 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

reaction was provided via polarity switching. This strategy commercially available with different specificities. Lectins are
allows exquisite control of the ratio of enzyme to substrate, the used for structural identification in CE by performing a
incubation time, and reagent mixing, while also providing a separation in the absence and then the presence of the lectin in
practical strategy to sustain the enzyme activity in a cost- the background electrolyte. When a structural match is present,
effective manner. These applications are significant because the lectin binds to the glycan, leading to a dramatic change in
linkage position holds potential to serve as a biomarker and CE the charge-to-size ratio. As shown in Figure 7B, this is observed
can complement other technologies that identify linkage with in the electropherogram as a peak that disappears. The
mass spectrometry through derivatization and fragmentation versatility of this approach was reported by Kinoshita, through
analyses,18,94 as well as separation via liquid chromatography95 separations performed in the absence and presence of a single
or ion mobility.18,47,94 lectin added to the background electrolyte in each run.97 A set
The advantages associated with in-capillary enzymolysis can of 14 different lectins were applied to N-glycans labeled with
be realized with a number of exoglycosidases. Yamagami et al. APTS or to milk oligosaccharides labeled with 2-aminobenzoic
demonstrated the utility of five different exoglycosidases: β- acid. These lectins, concanavalin A, wheat germ agglutinin,
galactosidase, α-mannosidase, β-acetylhexosaminidase, α-neu- Datura stramonium agglutinin, Aleuria aurantia lectin, Ulex
raminidase, and α-fucosidase for N-glycan identification.96 europaeus agglutinin, Ricinus communis agglutinin, soybean
These in-line enzyme reactions were accomplished with limited agglutinin, Maackia amurensis lectin, Pseudomonas aeruginosa
enzyme volumes within the separation time. Electrophoresis A lectin, Aspergillus oryzae lectin, Tulipa gesneriana agglutinin,
was used to drive the substrate through the enzyme zone with Crocus sativus lectin, Rhizopus stolonifer lectin, and Rhizopus
mixing or stopped flow incubation, which is called zero stolonifer, were purchased or were purified from plants by the
potential mixing. The method enabled determination of authors.
glycosidic linkage in N-glycans cleaved from different An alternative to filling the entire capillary with lectin is to
glycoproteins. confine the lectin to a zone within the capillary using a
4.2.2. Glycan Identification with the Use of Lectins. thermally reversible nanogel. This strategy enabled the use of
Lectins are glycan-binding proteins that recognize structural lectins and enzymes in series and was used to distinguish
features such as neuraminic acids or high mannose content. Galβ1−3GlcNAc from Galβ1−4GlcNAc linkages in N-
The specificity of lectins varies; for example, Sambucus nigra glycans.93 The advantage of this approach is that combinations
lectin binds only to α2−6 neuraminic acids while Maackia of lectins and enzymes can create unique specificity that is
Amurensis lectin binds only to α2−3 neuraminic acids. As difficult to realize with the lectins and enzymes that are
summarized in Table 2, many other lectins exist and are currently available. In the report by Holland et al, an enzyme
specific for β1−4 galactose residues was used in combination
Table 2. Examples of Commercial Lectins with Erythrina cristagalli lectin specific for β1−3,4 galactose
residues.93 Analysis of the N-glycan was accomplished with two
lectin class preferred glycan signaturea zones, with the first zone containing β1−4 galactosidase and
Fucose Binding Lectin the second containing Erythrina cristagalli lectin. The enzyme
Aleuria aurantia lectin (AAL) Fucα1−3,6 cleaved the terminal galactose residues with a Galβ1−4GlcNAc
Aspergillus oryzae lectin (AOL) Fucα1−6 (core) linkage, leaving any Galβ1−3GlcNAc linkage intact. The
Lotus tetragonolobus (LTA) Fucα1−3 Erythrina cristagalli lectin subsequently pulled down any N-
Ulex europaeus agglutinin I (UEA-I) Fucα1−2 glycan containing residual Galβ1−3GlcNAc linkages.
Galactose and N-Acetylgalactosamine Binding Lectin
4.3. Structural Verification with MS
Erythrina cristagalli lectin (ECL) Galβ1−4GlcNAc
Grif fonia simplicfolia isolectin B4(GSL Galβ1−3GlcNAc MS is used as a tool to complement CE separations for the
I−B4) identification of glycan structures. Matrix assisted laser
peanut agglutinin (PNA) Galβ1−3GalNAc desorption ionization55,59,98−101 and electrospray ioniza-
Phaseolus vulgaris leucoagglutinin terminal Gal tion91,98,102−105 are the most commonly employed ionization
(PHA-L) GlcNAcβ1−2,6Man (tri/ sources for N-glycan analysis. Matrix assisted laser desorption
tetraantennary)
ionization methods relying on exact mass59,98−100 and
Ricinus communis agglutinin (RCA) Galβ1−4GlcNAc, GalNAc
fragmentation55,101 have been reported for the determination
Soybean agglutinin (SBA) terminal Gal, GalNAc
of N-glycan structure. These matrix assisted laser desorption
N-Acetylneuraminic Acid Binding Lectins
ionization methods occur either entirely decoupled from the
Maackia amurensis lectin (MAL) NeuAcα2−3Galβ1−4GlcNAc
CE separation55,59,99,100 or, as shown in Figure 8, with the CE
Sambucus nigra agglutinin (SNA) NeuAcα2−6Gal(NAc)
instrument modified in order to spot eluent from the capillary
N-Acetyl Glucosamine Binding Lectin
onto a plate used for matrix assisted laser desorption
Datura stramonium lectin (DSL) GlcNAc
Phaseolus vulgaris erythroagglutinin bisecting GlcNAcβ1−4
ionization.98,101 Alternatively, the electrospray ionization
(PHA-E) terminal Gal (biantennary)
techniques occur almost exclusively in-line with the CE
Solanum tuberosum (STL) GlcNAc
separation.91,102−104 Sheathless interfaces that introduce ion
wheat germ agglutinin (WGA) GlcNAc
flow to complete the electrical circuit are fragile, but as shown
Glucose and Mannose Binding Lectins
in Figure 9, have been engineered to be easier for the user to
concanavalin A (Con A) αMan, αGlc
manipulate and operate.106 Glycan structure can be elucidated
Pisum sativum agglutinin (PSA) αMan, αGlc from electrospray ionization without fragmentation by employ-
ing mass deconvolution,98 a combination of exoglycosidases
a
Fucose (Fuc), galactose (Gal), N-acetylgalactosamine (GalNAc), and exact mass.91,103 However, several groups have demon-
glucose (Glc), N-acetylglucosamine (GlcNac), mannose (Man), N- strated the value of fragmentation with electrospray ionization
acetylneuraminic acid (NeuAc). for identifying N-glycan structure.104,105
7874 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

Figure 8. An instrument setup for off-line coupling of capillary

electrophoresis and matrix assisted laser desorption ionization mass
spectrometry (MALDI-MS). The capillary electrophoresis instrument
was modified to incorporate the automatic spotting device to deposit
the capillary eluent to the MALDI plate from the outlet vial of the
capillary electrophoresis instrument, and the matrix required for
MALDI detection was delivered from the inlet vial. From ref 101.
Copyright 2014 by John Wiley Sons, Inc. Reprinted by permission of
John Wiley & Sons, Inc.

Figure 9. A cross sectional view of a sheathless CE-ESI MS interface

including a porous tip in the front of the capillary outlet immersed in
the background electrolyte (BGE) inside the stainless steel cylinder. Figure 10. MS/MS spectrum of two glycans that overlapped in the
Reprinted with permission from ref 106. Copyright 2010 American capillary electrophoresis separation. The red line in the spectrum
Chemical Society. shows the signal (m/z) of the precursor ions selected for MS2 scan.
The observed Y-type fragment ions in the MS/MS spectrum indicate
A recent report based on a previous microfluidic study100 the identity of the monosaccharide that is lost. Reprinted from ref 104,
employed CE-MS electrospray ionization to reveal N-glycan Copyright 2017, with permission from Elsevier.
structures in serum samples using a combination of chemical
modification and fragmentation.104 In this paper, the micro- reporter, mass normalizer, and reactive functional groups.108
fluidic laser-induced fluorescence technique provided better The reactive functional groups (hydrazide or aminooxy
separation efficiency than the CE-MS; however, the advantage moieties) are attached to the reducing end of the N-glycans
of using the CE-MS method is that the information from intact through reductive amination. Both the mass reporter and mass
and fragment mass (m/z) enables the distinction of branched normalizer groups contain isotope elements (13C, D, or 15N),
N-glycans, which could not be identified based on the so that the total mass of the tandem mass tag between the
migration time of the standards alone due to comigration, as multiplexed labeling remains the same. Mass reporter ions are
shown in Figure 10. The sialylated (α2−6 and α2−3 linkages) generated by collision-induced dissociation along with N-glycan
N-glycans were derivatized with 4-(4,6-dimethoxy-1,3,5-triazin- fragments, and the relative abundance of each N-glycan is
2-yl)-4-methyl-morpholinium chloride following the produc- derived from the integrated area in extracted ion electrophero-
tion of amidated α2−6 linkages and lactones from α2−3 grams. The use of aminooxy tandem mass tag labeling for N-
linkages. As a result, α2−3 and α2−6 linkages were glycan analysis was performed using CE electrospray ionization
distinguished from one another based on differences in mass MS. Additionally, traveling wave ion mobility was incorporated
as well as the electrophoretic mobilities. Cross-ring fragmenta- after collision-induced dissociation as a second-dimension
tions generated from high energy collision induced dissociation separation based on gas-phase mobilities. This was utilized to
further enabled the technique to identify linkage and positional determine the relative abundance of N-glycan isomers through
isomers. As a result, 31 new N-glycan structures were improved resolution. To generate a high abundance of reporter
recognized with a total of 77 structures identified from serum ions in the collision-induced dissociation cell, a pseudo-MS3
samples, which was an improvement over the previous study method was applied.105 Instead of using collision-induced
which identified a total of 37 N-glycan structures.100 dissociation results (i.e., MS2 scan), both the intact N-glycan
Tandem mass tag, an isobaric tag, was designed to utilize and N-glycan fragments that contain tandem mass tag reporter
fragment ions derived from tandem mass spectrometry for ions are observed, as shown in Figure 11. The Y1 fragment (m/
quantitative analysis of peptides and proteins, as well as N- z = 523), which is composed of monosaccharide and tandem
glycans.105,107,108 Tandem mass tags are composed of the mass mass tag, was generated in the ion source by increasing the
7875 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

Figure 11. A representative MS/MS spectrum of a tandem mass tag (TMT) labeled glycan (m/z 779.8). The fragmentation of the TMT labeled
glycan is depicted in the structure above the spectrum. The reporter ions, used for quantification, are observed in the lower mass range of the
spectrum. Reprinted with permission from ref 105. Copyright 2015 American Chemical Society.

cone voltage from 30 to 100 V. Once Y1 fragments were The study by Hennig et al. utilized plasma samples from five
selected as the precursor ions, the mass spectra obtained from healthy male volunteers over periods ranging from 1.5 to 6
the MS2 scan reveals reporter ions in the absence of years resulting in 135 samples.88 Sampling intervals were more
interferences from N-glycan fragment ions. This is beneficial frequent during the first year. Lifestyle and health were noted 2
as the N-glycan fragments generally dominate the tandem mass days prior to blood sampling and correlated with changes in the
spectra and make it difficult to detect the reporter ion. The glycosylation profile. Plasma volumes of only 2 μL were used to
quantification of N-glycans, including both high mannose and obtain N-glycans that were separated using capillary gel
complex types, was demonstrated using the integrated areas of electrophoresis and an instrument with 16 capillaries in parallel.
the reporter ions in the MS2 scan generated from [M + Na + The N-glycan composition was characterized with the aid of
H]2+, [M + 2H]2+, and [M + 2H + K]3+ precursor ions. off-line exoglycosidase digestion (α2−3 and α2−3,6,8 sialidase;
α1−3,4 and α1−2,3,4,6 fucosidase; β1−4 galactosidase; α1−
5. APPLICATIONS 2,3,6 mannosidase; 1−2,3,4,6 N-acetyl glucosaminidase) of N-
glycans to identify the structure of the 31 most abundant
As described in this review, CE is a separation-based assay to
sialylated and asialylated peaks. The N-glycan assignment for
identify and quantify carbohydrates. Generally, the method is
each sample was assessed using a DNA internal standard with
adopted to address barriers in measurement technologies and
glyXtool for assignment by N-glycan migration time in the
then translated into a routine technique for monitoring
database, and the relative peak height proportions were
industrial or clinical processes. Several areas of high activity
calculated to transform migration data into an N-glycan
were noted from 2014 to 2017 and are reviewed here. Glycans
fingerprint. The reproducibility of the method was determined
were heavily studied during this review period and a strong
across many variables and the average variability in the relative
emphasis was apparent in biomarker discovery and biother-
peak height proportions were 5.4% RSD in overall (n = 10)
apeutic monitoring. This activity can be attributed to the
measurements with greater variability observed in low
separation power of CE and the ability to resolve complex
mixtures and to separate positional isomers based on subtle abundance peaks (11%) than for medium to high abundance
differences in hydrodynamic volume. This has a profound (3%). Except in cases of illness or injury, the variation within an
impact on biological therapeutics where glycosylation is linked individual was low throughout the time course of the study but
to product quality. Other areas, which are less prominent but was higher across individuals. Injury evoked an increase in
also warrant attention, include characterization of glycosami- sialylated N-glycans and a marginal increase in galactosylation.
noglycans, as well as analyses of carbohydrates in food and Illness, associated with allergic response, led to an increase in
plants. the amount of triantennary N-glycan accompanied by a
decrease in biantennary N-glycan composition. The authors
5.1. Biomarkers concluded that N-glycan biomarkers may be well suited as
5.1.1. Changes within and across Individuals. The personalized biomarkers, with changes in glycosylation
value of glycans as biomarkers has been established109 in the monitored in an individual routinely prior to illness.88
past 5 years and continues to advance steadily. The variability 5.1.2. Autoimmune Disorders. Glycosylation is strongly
associated with glycosylation as it occurs in the body must be linked to autoimmune disorders, and it has been postulated that
assessed to establish the value in monitoring glycosylation as a unique N-glycan signatures associated with specific motifs at a
biomarker for diseases. Hennig et al. noted a strong need to specific antibody glycosylation site serve to identify and
evaluate the stability of N-glycan profiles because most distinguish different autoimmune conditions.113 Current
biomarker studies involve measurements at a single time research focuses on changes in antibody glycosylation as a
point across disease states.88 The purpose of the work by means to provide insight into disease progression. Huang et al.
Hennig was to extend a prior study of individual variability used a combination of matrix assisted laser desorption
within a 1-week period110 and to build on prior reports that ionization time-of-flight MS for structural confirmation and
glycosylation is impacted by genetic background111 or age.112 CE for identification and quantification of N-glycan isomers
7876 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

associated with rheumatoid arthritis (n = 15 patients against n = The sialylated N-glycans from ovarian cancer patients were
15 controls).55 CE results centered on 11 N-glycans, which investigated using methylation of sialic acids to enhance the
demonstrated a decrease relative to healthy controls (p = separation.61 The benefit of modifying sialic acid residues, for
0.0001) in the galactosylation index, which is defined as the example, with methylation, is that chemical modification
fractional composition of galactosylated N-glycan to total N- eliminates the charge. The increased mass of each modified
glycan response.55 In a separate study, CE was used to evaluate residue leads to differences in mobility observed as slower
the responsiveness of IgG treatment for both rheumatoid migration times. This improves the peak resolution relative to
arthritis and Crohn’s disease to treatment with anti-TNFα.67 traditional CE approaches. Sialylated N-glycans derived from
The N-glycan was derived from IgG isolated in serum samples IgG in serum were separated using a labeled glycan ladder and
that were collected 2 weeks post-treatment. For rheumatoid quantified. Using the methylation strategy, researchers were
arthritis patients (n = 17), responding to the treatment (n = 11) able to successfully resolve biantennary N-glycan from
was associated with changes only in 3 low abundance monosialylated, fucosylated biantennary N-glycan, which
structures. Crohn’s disease patients responding to treatment enabled them to observe a significant increase in biantennary
(n = 14) exhibited changes in several low abundant and a single N-glycan in epithelial ovarian cancer patients (n = 5) relative to
high abundant N-glycan. One candidate biomarker from the the controls. A comparison of the results obtained for other N-
low abundance N-glycans was statistically significant (p = 0.01). glycans were similar with or without the methylation approach.
CE was also used in conjunction with matrix assisted laser Sialic acids containing N-glycans have been chemically
desorption ionization time-of-flight MS to profile HIV envelope modified via methylamidation to enhance peak resolution,
glycoprotein. Guttman et al. analyzed the N-glycan composition and the electrophoretic separations were capable of resolving
of the gp120 subunit of the HIV envelope glycoprotein.59 This N-glycans with different linkage positions.100 In a separate
subunit is of interest because it is highly (50% by mass) study, chemical modification of sialic acid residues was also
glycosylated. These researchers were able to demonstrate utilized in conjunction with microchip electrophoresis to
similarities among 3 recombinant gp120 samples (CM244, identify biomarkers in colon cancer.99 Modification of sialic
mother and infant) and then differences with a fourth sample acid residues led to a predictable change in the charge-to-size
(A244). Structural identification was possible using matrix ratio of N-glycans and allowed for a better distribution of the
assisted laser desorption ionization time-of-flight MS and a host N-glycans throughout the electrophoretic separation window.
of benchtop exoglycosidase reactions coupled with CE Differences in glycosylation among healthy controls (n = 20)
separations. A maltooligosaccharide ladder was used for peak and patients with colorectal cancer were evaluated following the
assignment across runs. first chemotherapy treatment (n = 26) and the third
5.1.3. Cancer. Excellent reviews have been published on the chemotherapy treatment (n = 16). The microchip electro-
relevance of glycosylation to cancer diagnosis and prognosis.114 phoresis yielded exceptional migration reproducibility (0.03%
Glycoconjugates are implicated in proliferation of malignant RSD), efficiency (700 000 theoretical plates), and separation
cells, migration, adhesion, and tumor evasion of the immune speed (135 s). Matrix assisted laser desorption ionization time-
system. A hallmark of cancer is a change in the glycosylation of-flight MS and electrophoresis were used as complementary
pattern in tumor tissue and serum.109,115,116 Mannose analytical tools, and both methods identified the same N-
branching,117 increased N-acetylglucosamine branching,9 bisect- glycans as potential biomarkers to distinguish healthy controls
ing N-acetylglucosamine,9 change in fucosylation9,118 or from cancer patients following different treatment time points.
polylactosamine,9,119 and increase in sialic acid content.9,109 These changes in the glycosylation profile were observed
Most work involves N-glycans derived from glycoproteins; between treatments, demonstrating the potential to use
although a few analyses of intact glycoproteins have been glycosylation profiles to stage colorectal cancer.
reported.120,121 Several glycosylation signatures are associated CE was used to identify two fucosylated N-glycans to
with many different types of cancer.109 distinguish light chain multiple myeloma from IgG myeloma
CE has recently been used to analyze N-glycans derived from from IgA myeloma using a set of 12 N-glycans collected from 2
IgG in serum as a method for diagnosing epithelial ovarian μL of serum from each patient.52 The relative abundance of
cancer.62 Schwedler et al. identified N-glycans using a standard each peak was used for statistical comparison and was
library derived from glycoproteins subject to exoglycosidase calculated as the sum of each peak to the cumulative peak
(benchtop) digestion and migration times evaluated with the area. In total, 167 samples were assessed from patients with
use of a GU migration time calibration. Through this effort 32 light chain multiple myeloma (n = 42), IgG myeloma (n = 42),
N-glycans were identified. When applied to a patient cohort of IgA myeloma (n = 41), and healthy controls (n = 42). In a
stage 3 or 4 epithelial ovarian cancer (n = 10) and healthy separate study,92 the same group evaluated N-glycans in serum
individuals (n = 5), researchers found significant differences in from a patient cohort with gastric cancer (n = 25) and gastric
increased N-glycan branching and fucosylation in the samples ulcer (n = 80) as compared to healthy controls (n = 139). The
from cancer patients. In a separate report, this research cohort study focused on fucosylation; 7 of the 9 N-glycans identified in
focused on changes in glycosylation attributed to acute phase the set were fucosylated. While specific fucosylated N-glycans
proteins to identify protein-specific differences in glycosylation decreased or increased, the total abundance of fucosylation
for epithelial ovarian cancer.121 Proteins extracted using reportedly decreased in gastric ulcer and in gastric cancer.
isoelectric focusing and two-dimensional slab gels were Other researchers also determined that differences in
identified with matrix assisted laser desorption ionization fucosylated N-glycans in urine, as well as differences in
time-of-flight MS and subsequently extracted, in order to triantennary structures, are potential biomarkers that comple-
analyze the N-glycans by CE. With a small patient cohort (n = 5 ment prostate specific antigen in order to better differentiate
cancer, n = 5 controls), several differences were identified patients with prostate cancer (n = 42) from those with benign
among 7 acute phase proteins. prostrate hyperplasia (n = 62).68
7877 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

5.1.4. Biomarker Studies of Cells. Others have also CE coupled with UV−visible absorbance detection or
worked to expand the role of glycosylation in development, electrospray ionization and MS were used to compare the
cellular differentiation, and aging. For instance, placental N- antibody-based drug cetuximab to a proposed generic
glycans have been profiled in an effort to obtain baseline data replacement.98 Cetuximab contains glycosylation sites in the
about glycosylation changes associated with the age of the antibody hinge region and near the Fab binding region. The
mother or the period of gestation.122 Both tissues cultured from glycosylation at these sites was distinguished using immuno-
animals and immortalized cells derived from tissue can be used globulin degrading enzyme to cleave the intact F(ab)2 region
to evaluate disease and the efficacy of treatment; however from the Fc chain. The protein fragments were separated and
fundamental questions remain about the suitability of these analyzed using a background electrolyte compatible with the
systems. Complex N-glycans and glycosaminoglycans were sheathless electrospray ionization-MS and a capillary covalently
monitored to provide insight into differences across an in vitro modified with hydroxypropylcellulose to suppress EOF. The
and in vivo model.76 Both N-glycans and glycosaminoglycans Fc2 fragment was resolved into multiple peaks, which
were isolated and labeled, although the glycosaminoglycans contained 2 terminal lysine variants and different glycosylation.
were first digested with Pronase then chondroitinase. When The F(ab)2 fragments were separated into multiple peaks each
analyzed by CE, carbohydrate profiles were different in cornea containing different N-glycans. Four different sialylated N-
tissue as compared to immortalized cornea cells (i.e., Statens glycan structures on some of the fragments were identified
Seruminstitut rabbit cornea cells). The reason for these because sialylated N-glycans cause both a mobility shift and
differences in N-glycan and glycosaminoglycan profiles was mass shift detectable by CE coupled to MS.
attributed to transition from epithelial to mesenchymal cells, CE coupled with sheathless electrospray ionization and MS
warranting caution when comparing results across the tissue was also used to quantitatively characterize Avonex, which is a
and immortalized cells derived from cornea tissue. recombinant interferon-β1.91 The protein was separated using a
Other approaches centered on models of cell differentiation. covalently cross-linked polyethyleneamine capillary and an
Thiesler et al. used CE in conjunction with several biochemical acidic background electrolyte under conditions of reversed
assays to demonstrate a change in the N-glycan profile in polarity. Of the 138 proteoforms, 55 were analyzed for
cultured cells used to model different stages of PMM2- deamidation, methionine loss, and glycosylation. The high
congenital disorder of glycosylation, a rare inherited disease.66 efficiency of the separation resolved isobaric positional isomers
These researchers focused on changes observed in high of sialic acid residues and polyLacNac. Bench top reactions with
mannose N-glycans in cells used to model cells from healthy exoglycosidases (galactosidase or sialidase) were conducted to
individuals as compared to phosphomannomutase 2-induced confirm these structures.
pluripotent stem cells developed to model the disorder. Many Gahoual et al. utilized CE coupled with sheathless electro-
biochemical methods were utilized to complement the CE. CE spray to compare two patented therapeutics with generic
separations of 22 N-glycans were transformed into fingerprints alternatives, called biosimilars.125 CE was used to analyze
using N-glycan standards derived from asialofetuin.66 Six glycopeptides from trastuzumab, cetuximab, and the biosimilar
abundant high-mannose-type N-glycans decreased in the candidates to establish glycosylation. Cetuximab has two sites
phosphomannomutase 2-induced pluripotent stem cells.66 In of glycosylation, and analysis of peptide fragments from the Fd
a separate study, changes in glycosylation were monitored domain within the antigen binding region established that the
during cellular differentiation of human induced pluripotent generic substitute did not contain α1−3 galactose residues
stem cells into cardiomyocytes using CE as well as MS.123 found in the innovator drug. This is important because α-
Migration time precision and normalization of peak area were galactose is a non-human structure that elicits an adverse
accomplished with an internal standard and GlyXtool software. immune response. A generic form of cetuximab lacking α-
Ten N-glycans changed across three time points (day 0, 7, and galactose is a better product than the original drug, potentially
15)123 and supported the findings of previous studies of making the generic form a new drug that can be patented.
cardiomyocyte differentiation. In addition, three unique N- A microchip electrophoresis separation with integrated
glycans previously not reported in these systems were found to nanoelectrospray ionization was coupled to MS to characterize
decrease after the initial (day 0) measurement.123 several features of an antibody−drug conjugate.126 The
5.2. Application to Biological Therapeutics approach demonstrated the ability to confirm the presence of
different glycosylation on the antibody. In a separate report, the
Sialylation on intact glycoproteins or glycopeptides is detected same chip-based nanoelectrospray system was used to assess
by CE because the negative charge changes the charge-to-size glycation on hemoglobin as a potential biomarker for diabetes
ratio and affects the mobility. This was demonstrated for a management.127
biological glycoprotein manufactured by Amgen.124 Proteins
were isolated using an isoelectric focusing separation prior to 5.3. Other Carbohydrates
CE with UV−visible absorbance detection. Using an acidic 5.3.1. Glycosaminoglycans. Several reports have appeared
background electrolyte, capillary coated with poly(vinyl during the time frame of this review on glycosaminoglycans,
alcohol), and reversed polarity, the product was separated which are linear polymers that contain repeating disaccharide
into 9 peaks of different sialic acid composition. The sialic acid units of uronic acid and hexosamine residues. The most widely
content was verified using a standard ninhydrin assay. Mapping discussed glycosaminoglycan is heparin. Heparin is used as an
of N-glycans was accomplished by enzymatically releasing N- anticoagulant, and monitoring the quality of heparin prior to
glycans, which were then analyzed using MS. The separation administering it to patients has become particularly important
could be used to evaluate protein products from different cell after the deaths of over 100 people, which were attributed to
clones, to monitor the optimization of the preparative scale the contamination of pharmaceutical heparin.128,129
purification of the therapeutic product, and finally to monitor Heparin is one of the most structurally complex
the product stability. glycosaminoglycans. Heparin has variable degrees of sulfation
7878 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

and alternating repeating units of α1−4 linked residues of L- distinguish weak and strong binding between glycosaminogly-
iduronic acid and N-,O-disulfated glucosamine, as shown in cans and apolipoproteins. An excellent discussion of the power
Figure 12. The R groups represent where the structure can be of CE, including different modes of separation, to elicit
information about glycosaminoglycans and proteins in general
has recently been published.137
5.3.2. Food and Plant Carbohydrate Analyses.
Carbohydrates relevant to food and plants pose several
challenges for analysis that have resulted in the development
of strategic techniques to overcome these barriers. These
carbohydrates are neutral and do not absorb well in the UV
range, which makes analysis using CE combined with UV
Figure 12. Schematic of heparin disaccharide. The repeating detection difficult. To facilitate the CE separation, mono- and
disaccharide is composed of L-iduronic acid and D-glucosamine joined disaccharides are commonly separated by employing a borate
by an α1−4 glycosidic linkage. The R group can be either sulfate or buffer, as has been demonstrated recently using samples such as
acetate and R′ and R″ can be either sulfate or hydrogen. caprine milk,138 honey,139 and the herb purslane.140 Boric acid
forms negatively charged complexes with diol groups, which
substituted with a sulfate, hydrogen, or acetate group. There are circumvents the issue of the carbohydrates being neutral and
eight possible combinations for heparin disaccharides depend- enables the separation of the mono- and disaccharides by
ing upon the substituent group. The sheer heterogeneity of charge-to-size ratio. These mono- and disaccharides can also be
glycosaminoglycan structures presents an analytical challenge separated by adjusting the pH of the background electrolyte to
that is addressed through analyses of disaccharide composition. a pH above 11. Using these extremely alkaline buffers causes
Analysis of even small glycosaminoglycan fragments is a deprotonation of the carbohydrates and provides a negative
daunting task and separation-based methods are still evolv- charge. The technique has been applied for the charge-to-size
ing.128,129 In an effort to address that need, a CE-MS method ratio separation of mono- and disaccharides in a wide variety of
providing label-free, rapid, and sensitive analysis to characterize food and plants, such as honey,141 breakfast cereals,142 juice and
sulfated disaccharides and low molecular weight heparins was nectar from fruits,143 and others.144−146 While most of these
developed employing both positive130 and negative modes.131 analyses rely on direct139,140,142,144 or indirect138,141,143,145
An electrokinetic pump-based interface was used to couple the detection by UV absorbance, one study demonstrated the use
CE separation to the analysis. Different degrees of sulfation on of capacitively coupled contactless conductivity detection
the disaccharide were separated due to variation in the carried coupled with electrospray ionization MS,146 which the authors
charges, and the identity was verified by exact mass. Both suggest can be used for the detection of neutral species that
bottom-up and top-down approaches were applied to reveal the may be overlooked in electrospray ionization MS due to poor
various sulfated disaccharides in Lovenox, a polycomponent ionization efficiency.
low molecular weight heparin used for anticoagulant treatment. More complex polysaccharides, such as amylopectin, found
Sulfated disaccharide structures in relatively low abundance in food and plants are typically analyzed using fluorophore
were identified with this method as a result of the 1000-fold assisted carbohydrate electrophoresis.45,147−150 For this techni-
enhancement in the limit of detection when the mass que, the polysaccharides are debranched using enzymes. The
spectrometer was utilized as compared to UV detection. polysaccharide fragments produced by debranching each
Twenty different types of low molecular weight heparins were contain a reducing end to which a fluorophore can be
identified with different characteristics with respect to sulfate, conjugated. This enables detection of the debranched
acetate, and 1,6 anhydrate groups.130 polysaccharides through laser-induced fluorescence. Debranch-
CE methods were also developed to profile and quantify ing also reduces the structure down to the linear chain,
other glycosaminoglycans, including heparan sulfate,132,133 removing the complication of changes in hydrodynamic volume
chondroitin sulfate,132−134 dermatan sulfate,133,134 hyalur- due to the branches when determining the degree of
onan,134 and keratin sulfate.133 Glycosaminoglycans are polymerization. Once derivatized with a fluorophore, the
typically first depolymerized using glycosaminoglycan carbohydrates are analyzed using a neutral coated capillary
lyases,132,133 which form disaccharides that are fluorescently under reverse polarity. This technique had been utilized to
labeled and separated based on the number of charged groups. quantify the polydispersity of amylopectin in food, a character-
One method achieved rapid profiling of up to 19 disaccharides istic that impacts the quality of rice,147 up to a degree of
in a single run. 133 CE techniques for profiling these polymerization of 160.148,150 While digestion is not necessary
glycosaminoglycans in urine have been demonstrated and for the carbohydrate analysis,45,147,148,150 one report demon-
hold the potential to be utilized for diagnosis of diseases in strates the applicability of this method to examine the ability for
newborns, since uronic acid glycosaminoglycans serve as a starch to be digested in the body by analyzing carbohydrate
biomarkers for various diseases.132 While depolymerization of profiles after performing an in vitro digestion.149
glycosaminoglycans is commonly performed, other studies
demonstrate the use of CE for the analysis of intact heparin, 6. EMERGING TECHNIQUES AND FUTURE
chondroitin sulfate, dermatan sulfate, and hyaluronan,134 as well DIRECTIONS
as K4 and K5 capsular polysaccharides that are chondroitin
sulfate and heparin starting materials.135 6.1. High Throughput Structural Characterization of
In addition to monitoring glycosaminoglycans in samples, Biological Therapeutics
CE has also been applied to study the binding interactions of The establishment of CE in so many diverse applications is a
glycosaminoglycans with apolipoproteins.136 Witos et al. testament to the importance of this method in glycosciences.
utilized a partial filling-affinity CE technique in order to Given the accelerating discoveries in glycosylation and the ever
7879 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

evolving need for new technologies, CE will play a more 6.2. Realizing the Full Power of Electrophoresis and MS
prominent role in this field. Certain aspects of this technology The benefit of more conclusive structural identification can be
are areas to watch for future growth. The first area of profound achieved if the strengths of MS and CE are leveraged. This is
impact will be as a tool to complement current18,47,94,95 and demonstrated in reports that utilize differences in mobilities to
emerging techniques151 to characterize and quantify positional distinguish linkage position102 or harness the MS to resolve
and linkage isomers for the exploding biological therapeutics peaks that comigrate102,104 in CE. A second area of growth to
market. Several collective innovations make this possible. watch is a growing use of commercial systems that couple CE
Significant progress has been achieved for migration time and MS. The commercialization of robust interfaces for end-
databases using polysaccharide or DNA ladders that yield users has dramatically increased the accessibility of the method.
unprecedented precision in time-based peak identification for These include the means to decouple the separation current
CE separations coupled with fluorescent detection. The sample from the electrospray process while avoiding dilution associated
preparation bottleneck is addressed with new tools for with the transfer of the nanoliter flow rates in the electro-
integrated and automated multistep processing. This stream- phoresis capillary.106 Improvements in the compatibility of
lined work flow for sample preparation begins with raw sample electrophoresis and MS, including new ionization strategies and
and ends with injection-ready glycans by automating antibody more flexibility in combining detection modalities, will increase
purification, deglycosylation, labeling, and sample clean up. the prevalence of CE in biomarker discovery. Growth in MS
With the easy generation of large batches of samples, citations in glycosciences have paralleled citation rates for
manufacturers of biologics have the power to easily and biomarker discovery. The current emphasis on analyses of
routinely monitor changes in glycosylation that can occur with glycopeptides and glycoproteins with CE-MS systems as
change in the cell culture conditions.10 This level of automated changes in both the glycosylation pattern as well as the
monitoring utilizing commercial robotics, as shown in Figure location of glycosylation may be more powerful in biomarker
13, will ultimately improve the quality of products delivered to specificity and sensitivity. Combining the unprecedented
consumers. separation power of CE, a migration database, and structural
determination would lead to a high level of certainty in
identification utilizing methods capable of high throughput
analyses.
A commercial microfluidic electrophoresis device designed
for MS is available (see Figure 14). The microfluidic device has

Figure 13. Photograph of the laboratory set up for the Biomek FXP
Laboratory Automation Workstation, which was used to automate
work flow for APTS labeling and purification of carbohydrates. The lab
materials required for this include a lid for the pipet box to reduce
evaporation (1), a 96-sample tray for a capillary electrophoresis
instrument (2), a Peltier shaker (3), 20 μL pipet tips (4), labeling
reagents and magnetic beads (5), a 96-well PCR plate on a magnetic
stand (6), 1000 μL pipet tips (7), and a 24-well plate for large volumes
of reagent (8). From ref 64. Copyright 2016 by SAGE Publications,
Reprinted by Permission of SAGE Publications, Inc.

The use of lectins and enzymes has become more common,

and commercial systems have been adapted to automatically Figure 14. Schematic of a commercially available microfluidic chip for
incubate and process exoglycosidase reactions for improved capillary electrophoresis−electrospray ionization−mass spectrometry.
linkage and monomer identification.152 The high cost and long The chip consists of sample reservoir S, background electrolyte
reaction times make integration of enzymatic processing less reservoir B, electrospray ionization pump P, and sample waste SW.
appealing but the potential of using in-line methods of enzyme The nanospray interface is integrated at the corner of the chip.
Adapted with permission from ref 107. Copyright 2017 American
processing are a promising solution.96 The recent discovery of Chemical Society.
materials to increase the enzyme lifetime from days to months
is fundamental to lowering the overall cost.77,93 A dramatic been utilized for glycoprotein and glycopeptide analy-
reduction in cost and time is realized when nanoliter reaction ses.107,127,153 This recently developed microfluidic device
volumes are used. Patterning separation capillaries with (ZipChip CE), which is surface modified to minimize protein
sequential enzyme cartridges is an effective step forward to adsorption and suppress EOF, integrates the electrospray
higher throughput screening with enzymes and is also interface into the microfluidic chip maintaining steady flow rate
invaluable to screening for the potential for enzymatic and electrospray ionization. Mass spectrometric compatible
remodeling of glycosylation. buffers were employed for the separation of glycopro-
7880 DOI: 10.1021/acs.chemrev.7b00669
Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

teins,126,127,153 glycopeptides,107 oligosaccharides,107 and Electroanalytical Research Group at the National Institute of
monosaccharides.107 The microfluidic devices have been Standards and Technology. She received her Ph.D. in Chemistry
utilized for separations of oligosaccharides and monosacchar- from the University of North Carolina at Chapel Hill under the
ides labeled with tandem mass tags, demonstrating the direction of Professor James Jorgenson. Through a National Research
structural identification and quantification observed with high Service Award she held a postdoctoral fellowship under the direction
throughput and automated benchtop instruments.107 of Professor Susan Lunte in the Department of Pharmaceutical
6.3. Future Directions Chemistry at the University of Kansas. Dr. Holland is the recipient of a
National Science Foundation Faculty Early Career Development
Researchers continue to tailor CE separations to meet the
award, has numerous publications in the field of separation chemistry,
challenges faced by the carbohydrate chemistry community. As
carbohydrate biomarkers gain more prominence, the technique and was elected to the position of executive board (2007−2012),
will be adapted further. Future applications may rely on more Chair-Elect (2012−2015), and Chair (2015−2017) of the American
portable methods of analyses based on microfluidic chips. Chemical Society Subdivision of Chromatography and Separation
These may utilize miniaturized detectors, contactless con- Chemistry. She holds a faculty position in the C. Eugene Bennett
ductivity detection, or fluorescence detection with light Department of Chemistry at West Virginia University, specializing in
emitting diodes and a CCD camera.154 Finally, as new microscale separations of biomolecules relevant to human health. She
carbohydrates continue to be identified and as researchers enjoys teaching instrumental analysis to undergraduate and graduate
strive to analyze highly complex mixtures, CE separations with students and mentoring the many outstanding researchers who have
higher peak capacity will gain more attention. studied separation science at WVU.

AUTHOR INFORMATION ACKNOWLEDGMENTS

Corresponding Author
This material is based upon work supported by NIH Grant No.
*E-mail: [email protected]. R01GM114330. C.L.C. acknowledges a National Science
ORCID Foundation IGERT fellowship, DGE no. 1144676.
Lisa A. Holland: 0000-0002-7534-6810
Notes REFERENCES
The authors declare no competing financial interest. (1) Pfrengle, F. Synthetic Plant Glycans. Curr. Opin. Chem. Biol.
2017, 40, 145−151.
Biographies (2) Shanker, S.; Hu, L.; Ramani, S.; Atmar, R. L.; Estes, M. K.;
Venkataram Prasad, B. V. Structural Features of Glycan Recognition
Grace (En-Tzu) Lu received both her B.S. and M.S. degrees in
among Viral Pathogens. Curr. Opin. Struct. Biol. 2017, 44, 211−218.
chemistry from National Taiwan University. Her research work (3) Kizuka, Y.; Kitazume, S.; Taniguchi, N. N-Glycan and Alzheimer’s
focused on glycan analyses using CE coupled with MS. In 2012, she Disease. Biochim. Biophys. Acta, Gen. Subj. 2017, 1861, 2447−2454.
joined the research group of Prof. Amanda J. Haes at the University of (4) Veillon, L.; Fakih, C.; Abou-El-Hassan, H.; Kobeissy, F.; Mechref,
Iowa, and in 2017, she completed her studies of CE analysis in Y. Glycosylation Changes in Brain Cancer. ACS Chem. Neurosci. 2018,
enzymatic reactions and small molecule detection using surface- 9, 51−72.
enhanced Raman scattering. She is currently a postdoctoral fellow in (5) Miura, Y.; Endo, T. Glycomics and Glycoproteomics Focused on
Professor Lisa A. Holland’s research group at West Virginia University. Aging and Age-Related Diseases  Glycans as a Potential Biomarker
Her research interests include developing analytical methods for for Physiological Alterations. Biochim. Biophys. Acta, Gen. Subj. 2016,
glycan structure elucidation using CE. 1860, 1608−1614.
(6) de Oliveira, M. A. L.; Porto, B. L. S.; de A. Bastos, C.; Sabarense,
Cassandra Crihfield received a B.S. degree in chemistry from Wheeling C. M.; Vaz, F. A. S.; Neves, L. N. O.; Duarte, L. M.; da S. Campos, N.;
Jesuit University in 2013. Cassandra is a Ph.D. candidate in analytical Chellini, P. R.; da Silva, P. H. F.; et al. Analysis of Amino Acids,
chemistry at West Virginia University in the Holland group. She is a Proteins, Carbohydrates and Lipids in Food by Capillary Electro-
current interdisciplinary graduate education and research traineeship migration Methods: A Review. Anal. Methods 2016, 8, 3649−3680.
(IGERT) fellow and former NanoSAFE fellow. Cassandra’s graduate (7) Yu, X.; Marshall, M. J. E.; Cragg, M. S.; Crispin, M. Improving
research has focused on developing enabling techniques based on CE Antibody-Based Cancer Therapeutics through Glycan Engineering.
for the detection and characterization of small molecules, oligosac- BioDrugs 2017, 31, 151−166.
(8) Gabius, H.-J. The Sugar Code: Why Glycans Are So Important.
charides, and proteins.
BioSystems 2018, 164, 102−111.
Srikanth Gattu has received his B.S. from Osmania University in 2009 (9) Kizuka, Y.; Taniguchi, N. Enzymes for N-Glycan Branching and
and then completed his M.S. from Vellore Institute of Technology in Their Genetic and Nongenetic Regulation in Cancer. Biomolecules
2011. He is currently a Ph.D. candidate in the research group of Prof. 2016, 6 (1−21), 25.
Lisa A Holland at West Virginia University. His research mainly (10) Batra, J.; Rathore, A. S. Glycosylation of Monoclonal Antibody
focuses on integrating microscale enzyme reactions and lectin Products: Current Status and Future Prospects. Biotechnol. Prog. 2016,
32, 1091−1102.
pulldowns using phospholipid assisted separations of biomolecules.
(11) Jefferis, R. Glyco-Engineering of Human IgG-Fc to Modulate
Lindsay Veltri is a senior undergraduate chemistry student at West Biologic Activities. Curr. Pharm. Biotechnol. 2016, 17, 1333−1347.
Virginia University. She is the recipient of a 2017 ACS Division of (12) Planinc, A.; Bones, J.; Dejaegher, B.; Van Antwerpen, P.;
Analytical Chemistry Undergraduate Award. Her academic interests Delporte, C. Glycan Characterization of Biopharmaceuticals: Updates
include the design and application of microfluidic devices for medical and Perspectives. Anal. Chim. Acta 2016, 921, 13−27.
diagnostics, drug discovery, and the development of nanomaterials for (13) Beyer, B.; Schuster, M.; Jungbauer, A.; Lingg, N. Micro-
biological detection. heterogeneity of Recombinant Antibodies: Analytics and Functional
Impact. Biotechnol. J. 2018, 13 (1−11), 1700476.
Lisa Holland received her B.S. degree in Chemistry from the (14) Mantovani, V.; Galeotti, F.; Maccari, F.; Volpi, N. Recent
University of Maryland at College Park, while working in the Advances in Capillary Electrophoresis Separation of Monosaccharides,

7881 DOI: 10.1021/acs.chemrev.7b00669

Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

Oligosaccharides, and Polysaccharides. Electrophoresis 2018, 39, 179− (34) Shajahan, A.; Heiss, C.; Ishihara, M.; Azadi, P. Glycomic and
189. Glycoproteomic Analysis of Glycoproteinsa Tutorial. Anal. Bioanal.
(15) Mantovani, V.; Galeotti, F.; Maccari, F.; Volpi, N. Recent Chem. 2017, 409, 4483−4505.
Advances on Separation and Characterization of Human Milk (35) Lindhorst Thisbe, K. Essentials of Carbohydrate Chemistry and
Oligosaccharides. Electrophoresis 2016, 37, 1514−1524. Biochemistry; John Wiley Sons: Chichester, U.K., 2003.
(16) Hajba, L.; Csanky, E.; Guttman, A. Liquid Phase Separation (36) Holland, L. A.; Chetwyn, N. P.; Perkins, M. D.; Lunte, S. M.
Methods for N-Glycosylation Analysis of Glycoproteins of Biomedical Capillary Electrophoresis in Pharmaceutical Analysis. Pharm. Res.
and Biopharmaceutical Interest. A Critical Review. Anal. Chim. Acta 1997, 14, 372−387.
2016, 943, 8−16. (37) Evangelista, R. A.; Liu, M.-S.; Chen, F.-T. A. Characterization of
(17) Fekete, S.; Guillarme, D.; Sandra, P.; Sandra, K. Chromato- 9-Aminopyrene-1,4,6-Trisulfonate Derivatized Sugars by Capillary
graphic, Electrophoretic, and Mass Spectrometric Methods for the Electrophoresis with Laser-Induced Fluorescence Detection. Anal.
Analytical Characterization of Protein Biopharmaceuticals. Anal. Chem. Chem. 1995, 67, 2239−2245.
2016, 88, 480−507. (38) Guttman, A. Analysis of Monosaccharide Composition by
(18) Gaunitz, S.; Nagy, G.; Pohl, N. L. B.; Novotny, M. V. Recent Capillary Electrophoresis. J. Chromatogr. A 1997, 763, 271−277.
Advances in the Analysis of Complex Glycoproteins. Anal. Chem. (39) Szabo, Z.; Guttman, A.; Rejtar, T.; Karger, B. L. Improved
2017, 89, 389−413. Sample Preparation Method for Glycan Analysis of Glycoproteins by
(19) Szekrényes, Á .; Park, S. S.; Santos, M.; Lew, C.; Jones, A.; Haxo, CE-LIF and CE-MS. Electrophoresis 2010, 31, 1389−1395.
T.; Kimzey, M.; Pourkaveh, S.; Szabó, Z.; Sosic, Z.; et al. Multi-Site N- (40) Váradi, C.; Lew, C.; Guttman, A. Rapid Magnetic Bead Based
Glycan Mapping Study 1: Capillary Electrophoresis − Laser Induced Sample Preparation for Automated and High Throughput N-Glycan
Fluorescence. mAbs 2016, 8, 56−64. Analysis of Therapeutic Antibodies. Anal. Chem. 2014, 86, 5682−5687.
(20) https://sciex.com/products/standards-and-reagents/fast-glycan- (41) Oda, R. P.; Landers, J. P. In Handbook of Capillary
analysis-and-labeling-for-the-pa-800-plus, accessed November 01, Electrophoresis, 2nd ed.; Landers, J. P., Ed.; CRC Press: Boca Raton,
2017. FL, 1997; Chapter 1.
(21) https://www.thermofisher.com/order/catalog/product/ (42) Guttman, A.; Cooke, N.; Starr, C. M. Capillary Electrophoresis
A28676, accessed November 1, 2017. Separation of Oligosaccharides: I. Effect of Operational Variables.
(22) https://prozyme.com/products/gp24ng-apts, accessed Novem- Electrophoresis 1994, 15, 1518−1522.
ber 01, 2017. (43) Barnes, J.; Tian, L.; Loftis, J.; Hiznay, J.; Comhair, S.; Lauer, M.;
(23) https://www.ludger.com/products/glycan_labeling_kits.php, Dweik, R. Isolation and Analysis of Sugar Nucleotides Using Solid
accessed February 17, 2018. Phase Extraction and Fluorophore Assisted Carbohydrate Electro-
(24) www.waters.com/waters/en_CA/GlycoWorks-RapiFluor-MS- phoresis. MethodsX 2016, 3, 251−260.
N-Glycan-Kit/nav.htm?locale=en_CA&cid=134828150, accessed Feb- (44) Karousou, E.; Asimakopoulou, A.; Monti, L.; Zafeiropoulou, V.;
ruary 17, 2018. Afratis, N.; Gartaganis, P.; Rossi, A.; Passi, A.; Karamanos, N. K. Face
(25) Ecker, D. M.; Jones, S. D.; Levine, H. L. The Therapeutic Analysis as a Fast and Reliable Methodology to Monitor the Sulfation
Monoclonal Antibody Market. mAbs 2015, 7, 9−14. and Total Amount of Chondroitin Sulfate in Biological Samples of
(26) Jorgenson, J. W.; Lukacs, K. D. Capillary Zone Electrophoresis. Clinical Importance. Molecules 2014, 19, 7959−7980.
Science 1983, 222, 266−272. (45) Kuang, Q.; Xu, J.; Wang, K.; Zhou, S.; Liu, X. Structure and
(27) Reusch, D.; Haberger, M.; Kailich, T.; Heidenreich, A.-K.; Digestion of Hybrid Indica Rice Starch and Its Biosynthesis. Int. J. Biol.
Kampe, M.; Bulau, P.; Wuhrer, M. High-Throughput Glycosylation Macromol. 2016, 93, 402−407.
Analysis of Therapeutic Immunoglobulin G by Capillary Gel (46) Vreeker, G.; Wuhrer, M. Reversed-Phase Separation Methods
Electrophoresis Using a DNA Analyzer. mAbs 2014, 6, 185−196. for Glycan Analysis. Anal. Bioanal. Chem. 2017, 409, 359−378.
(28) Feng, H.-t.; Lim, S.; Laserna, A. K. C.; Li, P.; Yin, X.; Simsek, E.; (47) Hofmann, J.; Pagel, K. Glycan Analysis by Ion Mobility−Mass
Khan, S. H.; Chen, S.-M.; Li, S. F. Y. High Throughput Human Plasma Spectrometry. Angew. Chem., Int. Ed. 2017, 56, 8342−8349.
N-Glycan Analysis Using DNA Analyzer and Multivariate Analysis for (48) Geissner, A.; Seeberger, P. H. Glycan Arrays: From Basic
Biomarker Discovery. Anal. Chim. Acta 2017, 995, 106−113. Biochemical Research to Bioanalytical and Biomedical Applications.
(29) Mahan, A. E.; Tedesco, J.; Dionne, K.; Baruah, K.; Cheng, H. D.; Annu. Rev. Anal. Chem. 2016, 9, 223−247.
De Jager, P. L.; Barouch, D. H.; Suscovich, T.; Ackerman, M.; Crispin, (49) Reusch, D.; Haberger, M.; Maier, B.; Maier, M.; Kloseck, R.;
M.; Alter, G. A Method for High-Throughput, Sensitive Analysis of Zimmermann, B.; Hook, M.; Szabo, Z.; Tep, S.; Wegstein, J.; Alt, N.;
IgG Fc and Fab Glycosylation by Capillary Electrophoresis. J. Immunol. Bulau, P.; Wuhrer, M. Comparison of Methods for the Analysis of
Methods 2015, 417, 34−44. Therapeutic Immunoglobulin G Fc-Glycosylation Profiles-Part 1:
(30) Huffman, J. E.; Pučić-Baković, M.; Klarić, L.; Hennig, R.; Separation-Based Methods. mAbs 2015, 7, 167−179.
Selman, M. H. J.; Vučković, F.; Novokmet, M.; Krištić, J.; Borowiak, (50) Adamczyk, B.; Tharmalingam-Jaikaran, T.; Schomberg, M.;
M.; Muth, T.; et al. Comparative Performance of Four Methods for Szekrényes, Á .; Kelly, R. M.; Karlsson, N. G.; Guttman, A.; Rudd, P.
High-Throughput Glycosylation Analysis of Immunoglobulin G in M. Comparison of Separation Techniques for the Elucidation of IgG
Genetic and Epidemiological Research. Mol. Cell. Proteomics 2014, 13, N-Glycans Pooled from Healthy Mammalian Species. Carbohydr. Res.
1598−1610. 2014, 389, 174−185.
(31) Feng, H.-t.; Li, P.; Rui, G.; Stray, J.; Khan, S.; Chen, S.-M.; Li, S. (51) Danyluk, H. J.; Shum, L. K.; Zandberg, W. F. In Protein-
F. Y. Multiplexing N-Glycan Analysis by DNA Analyzer. Electrophoresis Carbohydrate Interactions: Methods and Protocols; Abbott, D. W.,
2017, 38, 1788−1799. Lammerts van Bueren, A., Eds.; Humana Press: New York, NY, 2017,
(32) Kovács, Z.; Szarka, M.; Szigeti, M.; Guttman, A. Separation 1588, 223−236.
Window Dependent Multiple Injection (Swdmi) for Large Scale (52) Chen, J.; Fang, M.; Zhao, Y.-P.; Yi, C.-H.; Ji, J.; Cheng, C.;
Analysis of Therapeutic Antibody N-Glycans. J. Pharm. Biomed. Anal. Wang, M.-M.; Gu, X.; Sun, Q.-S.; Chen, X.-L.; Gao, C.-F. Serum N-
2016, 128, 367−370. Glycans: A New Diagnostic Biomarker for Light Chain Multiple
(33) Stanley, P.; Schachter, H.; Taniguchi, N. In Essentials of Myeloma. PLoS One 2015, 10, No. e0127022.
Glycobiology, 2nd ed.; Varki, A., Cummings, R. D., Esko, J. D., et al., (53) Donczo, B.; Szigeti, M.; Ostoros, G.; Gacs, A.; Tovari, J.;
Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor Guttman, A. N-Glycosylation Analysis of Formalin Fixed Paraffin
(NY), 2009; Chapter 8. Available from: https://www.ncbi.nlm.nih. Embedded Samples by Capillary Electrophoresis. Electrophoresis 2016,
gov/books/NBK1917/. 37, 2292−2296.

7882 DOI: 10.1021/acs.chemrev.7b00669

Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

(54) Guttman, A.; Kerekgyarto, M.; Jarvas, G. Effect of Separation (70) Jooß, K.; Sommer, J.; Bunz, S.-C.; Neusüß, C. In-Line SPE-CE
Temperature on Structure Specific Glycan Migration in Capillary Using a Fritless Bead String DesignApplication for the Analysis of
Electrophoresis. Anal. Chem. 2015, 87, 11630−11634. Organic Sulfonates Including Inline SPE-CE-MS for APTS-Labeled
(55) Huang, C.; Liu, Y.; Wu, H.; Sun, D.; Li, Y. Characterization of Glycans. Electrophoresis 2014, 35, 1236−1243.
IgG Glycosylation in Rheumatoid Arthritis Patients by MALDI-TOF- (71) Laukens, B.; De Wachter, C.; Callewaert, N. In Glyco-
MSn and Capillary Electrophoresis. Anal. Bioanal. Chem. 2017, 409, Engineering: Methods and Protocols; Castilho, A., Ed.; Humana Press:
3731−3739. New York, NY, 2015, 1321, 103−122.
(56) Jarvas, G.; Szigeti, M.; Guttman, A. GUcal: An Integrated (72) O’Regan, N. L.; Steinfelder, S.; Schwedler, C.; Rao, G. B.;
Application for Capillary Electrophoresis Based Glycan Analysis. Srikantam, A.; Blanchard, V.; Hartmann, S. Filariasis Asymptomatically
Electrophoresis 2015, 36, 3094−3096. Infected Donors Have Lower Levels of Disialylated IgG Compared to
(57) Kovács, Z.; Papp, G.; Horváth, H.; Joó, F.; Guttman, A. A Novel Endemic Normals. Parasite Immunol. 2014, 36, 713−720.
Carbohydrate Labeling Method Utilizing Transfer Hydrogenation- (73) Wang, M.; Fang, M.; Zhu, J.; Feng, H.; Warner, E.; Yi, C.; Ji, J.;
Mediated Reductive Amination. J. Pharm. Biomed. Anal. 2017, 142, Gu, X.; Gao, C. Serum N-Glycans Outperform CA19−9 in Diagnosis
324−327. of Extrahepatic Cholangiocarcinoma. Electrophoresis 2017, 38, 2749−
(58) Donczo, B.; Szarka, M.; Tovari, J.; Ostoros, G.; Csanky, E.; 2756.
Guttman, A. Molecular Glycopathology by Capillary Electrophoresis: (74) Ruhaak, L. R.; Zauner, G.; Huhn, C.; Bruggink, C.; Deelder, A.
Analysis of the N-Glycome of Formalin-Fixed Paraffin-Embedded M.; Wuhrer, M. Glycan Labeling Strategies and Their Use in
Mouse Tissue Samples. Electrophoresis 2017, 38, 1602−1608. Identification and Quantification. Anal. Bioanal. Chem. 2010, 397,
(59) Guttman, M.; Váradi, C.; Lee, K. K.; Guttman, A. Comparative 3457−3481.
Glycoprofiling of Hiv Gp120 Immunogens by Capillary Electro- (75) Uematsu, R.; Furukawa, J.-i.; Nakagawa, H.; Shinohara, Y.;
phoresis and MALDI Mass Spectrometry. Electrophoresis 2015, 36, Deguchi, K.; Monde, K.; Nishimura, S.-I. High Throughput
1305−1313. Quantitative Glycomics and Glycoform-Focused Proteomics of
(60) Meininger, M.; Stepath, M.; Hennig, R.; Cajic, S.; Rapp, E.; Murine Dermis and Epidermis. Mol. Cell. Proteomics 2005, 4, 1977−
Rotering, H.; Wolff, M. W.; Reichl, U. Sialic Acid-Specific Affinity 1989.
Chromatography for the Separation of Erythropoietin Glycoforms (76) Iwatsuka, K.; Iwamoto, H.; Kinoshita, M.; Inada, K.; Yasueda, S.-
Using Serotonin as a Ligand. J. Chromatogr. B: Anal. Technol. Biomed. i.; Kakehi, K. Comparative Studies of N-Glycans and Glycosamino-
Life Sci. 2016, 1012, 193−203. glycans Present in SIRC (Statens Seruminstitut Rabbit Cornea) Cells
(61) Schwedler, C.; Kaup, M.; Petzold, D.; Hoppe, B.; Braicu, E. I.; and Corneal Epithelial Cells from Rabbit Eyes. Curr. Eye Res. 2014, 39,
Sehouli, J.; Ehlers, M.; Berger, M.; Tauber, R.; Blanchard, V. Sialic Acid 686−694.
Methylation Refines Capillary Electrophoresis Laser-Induced Fluo- (77) Gattu, S.; Crihfield, C. L.; Holland, L. A. Microscale
rescence Analyses of Immunoglobulin G N-Glycans of Ovarian Cancer Measurements of Michaelis−Menten Constants of Neuraminidase
with Nanogel Capillary Electrophoresis for the Determination of the
Patients. Electrophoresis 2014, 35, 1025−1031.
(62) Schwedler, C.; Kaup, M.; Weiz, S.; Hoppe, M.; Braicu, E. I.; Sialic Acid Linkage. Anal. Chem. 2017, 89, 929−936.
(78) Váradi, C.; Mittermayr, S.; Millán-Martín, S.; Bones, J.
Sehouli, J.; Hoppe, B.; Tauber, R.; Berger, M.; Blanchard, V.
Quantitative Twoplex Glycan Analysis Using 12C6 and 13C6 Stable
Identification of 34 N-Glycan Isomers in Human Serum by Capillary
Isotope 2-Aminobenzoic Acid Labelling and Capillary Electrophoresis
Electrophoresis Coupled with Laser-Induced Fluorescence Allows
Mass Spectrometry. Anal. Bioanal. Chem. 2016, 408, 8691−8700.
Improving Glycan Biomarker Discovery. Anal. Bioanal. Chem. 2014,
(79) Yamamoto, S.; Nagai, E.; Asada, Y.; Kinoshita, M.; Suzuki, S. A
406, 7185−7193. Rapid and Highly Sensitive Microchip Electrophoresis of Mono- and
(63) Szekrényes, Á .; Partyka, J.; Varadi, C.; Krenkova, J.; Foret, F.;
Mucin-Type Oligosaccharides Labeled with 7-Amino-4-Methylcou-
Guttman, A.In Microchip Capillary Electrophoresis Protocols; Van marin. Anal. Bioanal. Chem. 2015, 407, 1499−1503.
Schepdael, A., Ed.; Humana Press: New York, NY, 2015, 1274, (80) Yodoshi, M.; Ikeda, N.; Yamaguchi, N.; Nagata, M.; Nishida, N.;
183−195. Kakehi, K.; Hayakawa, T.; Suzuki, S. A Novel Condition for Capillary
(64) Szigeti, M.; Lew, C.; Roby, K.; Guttman, A. Fully Automated Electrophoretic Analysis of Reductively Aminated Saccharides without
Sample Preparation for Ultrafast N-Glycosylation Analysis of Antibody Removal of Excess Reagents. Electrophoresis 2013, 34, 3198−3205.
Therapeutics. J. Lab. Autom. 2016, 21, 281−286. (81) Song, X.; Ju, H.; Lasanajak, Y.; Kudelka, M. R.; Smith, D. F.;
(65) Szigeti, M.; Bondar, J.; Gjerde, D.; Keresztessy, Z.; Szekrenyes, Cummings, R. D. Oxidative Release of Natural Glycans for Functional
A.; Guttman, A. Rapid N-Glycan Release from Glycoproteins Using Glycomics. Nat. Nat. Methods 2016, 13, 528−534.
Immobilized PNGase F Microcolumns. J. Chromatogr. B: Anal. (82) Mittermayr, S.; Guttman, A. Influence of Molecular
Technol. Biomed. Life Sci. 2016, 1032, 139−143. Configuration and Conformation on the Electromigration of
(66) Thiesler, C. T.; Cajic, S.; Hoffmann, D.; Thiel, C.; van Diepen, Oligosaccharides in Narrow Bore Capillaries. Electrophoresis 2012,
L.; Hennig, R.; Sgodda, M.; Weiβmann, R.; Reichl, U.; Steinemann, 33, 1000−1007.
D.; et al. Glycomic Characterization of Induced Pluripotent Stem Cells (83) Guttman, A.; Chen, F.-T. A.; Evangelista, R. A. Separation of 1-
Derived from a Patient Suffering from Phosphomannomutase 2 Aminopyrene-3,6,8-Trisulfonic-Labeled Asparagine-Linked Fetuin
Congenital Disorder of Glycosylation (PMM2-CDG). Mol. Cell. Glycans by Capillary Electrophoresis. Electrophoresis 1996, 17, 412−
Proteomics 2016, 15, 1435−1452. 417.
(67) Váradi, C.; Hóllo, Z.; Póliska, S.; Nagy, L.; Szekanecz, Z.; (84) Guttman, A.; Herrick, S. Effect of the Quantity and Linkage
Váncsa, A.; Palatka, K.; Guttman, A. Combination of IgG N-Glycomics Position of Mannose(α1,2) Residues in Capillary Gel Electrophoresis
and Corresponding Transcriptomics Data to Identify Anti-TNF-α of High-Mannose-Type Oligosaccharides. Anal. Biochem. 1996, 235,
Treatment Responders in Inflammatory Diseases. Electrophoresis 2015, 236−239.
36, 1330−1335. (85) Jarvas, G.; Szigeti, M.; Chapman, J.; Guttman, A. Triple-Internal
(68) Vermassen, T.; Van Praet, C.; Vanderschaeghe, D.; Maenhout, Standard Based Glycan Structural Assignment Method for Capillary
T.; Lumen, N.; Callewaert, N.; Hoebeke, P.; Van Belle, S.; Rottey, S.; Electrophoresis Analysis of Carbohydrates. Anal. Chem. 2016, 88,
Delanghe, J. Capillary Electrophoresis of Urinary Prostate Glyco- 11364−11367.
proteins Assists in the Diagnosis of Prostate Cancer. Electrophoresis (86) https://lendulet.uni-pannon.hu/index.php/tools, accessed Oc-
2014, 35, 1017−1024. tober 30, 2017.
(69) Hennig, R.; Rapp, E.; Kottler, R.; Cajic, S.; Borowiak, M.; Reichl, (87) Szigeti, M.; Guttman, A. High-Resolution Glycan Analysis by
U. In Carbohydrate-Based Vaccines: Methods and Protocols; Lepenies, B., Temperature Gradient Capillary Electrophoresis. Anal. Chem. 2017,
Ed.; Humana Press: New York, NY, 2015, 1331, 123−143. 89, 2201−2204.

7883 DOI: 10.1021/acs.chemrev.7b00669

Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

(88) Hennig, R.; Cajic, S.; Borowiak, M.; Hoffmann, M.; Kottler, R.; (105) Zhong, X.; Chen, Z.; Snovida, S.; Liu, Y.; Rogers, J. C.; Li, L.
Reichl, U.; Rapp, E. Towards Personalized Diagnostics via Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry
Longitudinal Study of the Human Plasma N-Glycome. Biochim. for Quantitative Analysis of Glycans Labeled with Multiplex Carbonyl-
Biophys. Acta, Gen. Subj. 2016, 1860, 1728−1738. Reactive Tandem Mass Tags. Anal. Chem. 2015, 87, 6527−6534.
(89) Behne, A.; Muth, T.; Borowiak, M.; Reichl, U.; Rapp, E. (106) Busnel, J.-M.; Schoenmaker, B.; Ramautar, R.; Carrasco-
glyXalign: High-Throughput Migration Time Alignment Preprocess- Pancorbo, A.; Ratnayake, C.; Feitelson, J. S.; Chapman, J. D.; Deelder,
ing of Electrophoretic Data Retrieved via Multiplexed Capillary Gel A. M.; Mayboroda, O. A. High Capacity Capillary Electrophoresis-
Electrophoresis with Laser-Induced Fluorescence Detection-Based Electrospray Ionization Mass Spectrometry: Coupling a Porous
Glycoprofiling. Electrophoresis 2013, 34, 2311−2315. Sheathless Interface with Transient-Isotachophoresis. Anal. Chem.
(90) Guttman, A.; Ulfelder, K. W. Exoglycosidase Matrix-Mediated 2010, 82, 9476−9483.
Sequencing of a Complex Glycan Pool by Capillary Electrophoresis. J. (107) Khatri, K.; Klein, J. A.; Haserick, J. R.; Leon, D. R.; Costello, C.
Chromatogr. A 1997, 781, 547−554. E.; McComb, M. E.; Zaia, J. Microfluidic Capillary Electrophoresis−
(91) Bush, D. R.; Zang, L.; Belov, A. M.; Ivanov, A. R.; Karger, B. L. Mass Spectrometry for Analysis of Monosaccharides, Oligosaccharides,
High Resolution CZE-MS Quantitative Characterization of Intact and Glycopeptides. Anal. Chem. 2017, 89, 6645−6655.
Biopharmaceutical Proteins: Proteoforms of Interferon-β1. Anal. (108) Hahne, H.; Neubert, P.; Kuhn, K.; Etienne, C.; Bomgarden, R.;
Chem. 2016, 88, 1138−1146. Rogers, J. C.; Kuster, B. Carbonyl-Reactive Tandem Mass Tags for the
(92) Zhao, Y.-P.; Xu, X.-Y.; Fang, M.; Wang, H.; You, Q.; Yi, C.-H.; Proteome-Wide Quantification of N-Linked Glycans. Anal. Chem.
Ji, J.; Gu, X.; Zhou, P.-T.; Cheng, C.; Gao, C.-F. Decreased Core- 2012, 84, 3716−3724.
Fucosylation Contributes to Malignancy in Gastric Cancer. PLoS One (109) Dube, D. H.; Bertozzi, C. R. Glycans in Cancer and
2014, 9, No. e94536. Inflammation - Potential for Therapeutics and Diagnostics. Nat. Rev.
(93) Holland, L. A.; Gattu, S.; Crihfield, C. L.; Bwanali, L. Capillary Drug Discovery 2005, 4, 477−488.
Electrophoresis with Stationary Nanogel Zones of Galactosidase and (110) Gornik, O.; Wagner, J.; Pučić, M.; Knežević, A.; Redžić, I.;
Erythrina cristagalli Lectin for the Determination of β(1−3)-Linked Lauc, G. Stability of N-Glycan Profiles in Human Plasma. Glycobiology
Galactose in Glycans. J. Chromatogr. A 2017, 1523, 90−96. 2009, 19, 1547−1553.
(94) Alley, W. R.; Mann, B. F.; Novotny, M. V. High-Sensitivity (111) Pučić, M.; Knežević, A.; Vidič, J.; Adamczyk, B.; Novokmet,
Analytical Approaches for the Structural Characterization of M.; Polašek, O.; Gornik, O.; Šupraha-Goreta, S.; Wormald, M. R.;
Glycoproteins. Chem. Rev. 2013, 113, 2668−2732. Redžić, I.; et al. High Throughput Isolation and Glycosylation Analysis
(95) Veillon, L.; Huang, Y.; Peng, W.; Dong, X.; Cho, B. G.; Mechref, of IgG−Variability and Heritability of the IgG Glycome in Three
Y. Characterization of Isomeric Glycan Structures by LC-MS/MS.
Isolated Human Populations. Mol. Cell. Proteomics 2011, 10 (1−15),
Electrophoresis 2017, 38, 2100−2114.
M111.010090.
(96) Yamagami, M.; Matsui, Y.; Hayakawa, T.; Yamamoto, S.;
(112) Vanhooren, V.; Dewaele, S.; Libert, C.; Engelborghs, S.; De
Kinoshita, M.; Suzuki, S. Plug-Plug Kinetic Capillary Electrophoresis
Deyn, P. P.; Toussaint, O.; Debacq-Chainiaux, F.; Poulain, M.;
for in-Capillary Exoglycosidase Digestion as a Profiling Tool for the
Glupczynski, Y.; Franceschi, C.; et al. Serum N-Glycan Profile Shift
Analysis of Glycoprotein Glycans. J. Chromatogr. A 2017, 1496, 157−
During Human Ageing. Exp. Gerontol. 2010, 45, 738−743.
162.
(113) Maverakis, E.; Kim, K.; Shimoda, M.; Gershwin, M. E.; Patel,
(97) Kinoshita, M.; Kakehi, K. Capillary-Based Lectin Affinity
Electrophoresis for Interaction Analysis between Lectins and Glycans. F.; Wilken, R.; Raychaudhuri, S.; Ruhaak, L. R.; Lebrilla, C. B. Glycans
Methods Mol. Biol. 2014, 1200, 131−146. in the Immune System and the Altered Glycan Theory of
(98) Biacchi, M.; Gahoual, R.; Said, N.; Beck, A.; Leize-Wagner, E.; Autoimmunity: A Critical Review. J. Autoimmun. 2015, 57, 1−13.
François, Y.-N. Glycoform Separation and Characterization of (114) Adamczyk, B.; Tharmalingam, T.; Rudd, P. M. Glycans as
Cetuximab Variants by Middle-up Off-Line Capillary Zone Electro- Cancer Biomarkers. Biochim. Biophys. Acta, Gen. Subj. 2012, 1820,
phoresis-Uv/Electrospray Ionization-Ms. Anal. Chem. 2015, 87, 6240− 1347−1353.
6250. (115) Peracaula, R.; Barrabés, S.; Sarrats, A.; Rudd, P. M.; de Llorens,
(99) Snyder, C. M.; Alley, W. R.; Campos, M. I.; Svoboda, M.; Goetz, R. Altered Glycosylation in Tumours Focused to Cancer Diagnosis.
J. A.; Vasseur, J. A.; Jacobson, S. C.; Novotny, M. V. Complementary Dis. Dis. Markers 2008, 25, 207−218.
Glycomic Analyses of Sera Derived from Colorectal Cancer Patients (116) Fuster, M. M.; Esko, J. D. The Sweet and Sour of Cancer:
by Maldi-Tof-Ms and Microchip Electrophoresis. Anal. Chem. 2016, Glycans as Novel Therapeutic Targets. Nat. Rev. Cancer 2005, 5, 526−
88, 9597−9605. 542.
(100) Mitra, I.; Snyder, C. M.; Zhou, X.; Campos, M. I.; Alley, W. R.; (117) de Leoz, M. L. A.; Young, L. J. T.; An, H. J.; Kronewitter, S. R.;
Novotny, M. V.; Jacobson, S. C. Structural Characterization of Serum Kim, J.; Miyamoto, S.; Borowsky, A. D.; Chew, H. K.; Lebrilla, C. B.
N-Glycans by Methylamidation, Fluorescent Labeling, and Analysis by High-Mannose Glycans Are Elevated During Breast Cancer Pro-
Microchip Electrophoresis. Anal. Chem. 2016, 88, 8965−8971. gression. Mol. Cell. Proteomics 2011, 10, M110.002717.
(101) Biacchi, M.; Bhajun, R.; Saïd, N.; Beck, A.; François, Y. N.; (118) Miyoshi, E.; Moriwaki, K.; Nakagawa, T. Biological Function of
Leize-Wagner, E. Analysis of Monoclonal Antibody by a Novel CE- Fucosylation in Cancer Biology. J. Biochem. 2008, 143, 725−729.
UV/MALDI-MS Interface. Electrophoresis 2014, 35, 2986−2995. (119) Dennis, J. W.; Granovsky, M.; Warren, C. E. Glycoprotein
(102) Kammeijer, G. S. M.; Jansen, B. C.; Kohler, I.; Heemskerk, A. Glycosylation and Cancer Progression. Biochim. Biophys. Acta, Gen.
A. M.; Mayboroda, O. A.; Hensbergen, P. J.; Schappler, J.; Wuhrer, M. Subj. 1999, 1473, 21−34.
Sialic Acid Linkage Differentiation of Glycopeptides Using Capillary (120) Balmaña, M.; Giménez, E.; Puerta, A.; Llop, E.; Figueras, J.;
Electrophoresis − Electrospray Ionization − Mass Spectrometry. Sci. Fort, E.; Sanz-Nebot, V.; de Bolós, C.; Rizzi, A.; Barrabés, S.; et al.
Rep. 2017, 7, 3733. Increased α1−3 Fucosylation of α-1-Acid Glycoprotein (AGP) in
(103) Jayo, R. G.; Thaysen-Andersen, M.; Lindenburg, P. W.; Pancreatic Cancer. J. Proteomics 2016, 132, 144−154.
Haselberg, R.; Hankemeier, T.; Ramautar, R.; Chen, D. D. Y. Simple (121) Weiz, S.; Wieczorek, M.; Schwedler, C.; Kaup, M.; Braicu, E. I.;
Capillary Electrophoresis−Mass Spectrometry Method for Complex Sehouli, J.; Tauber, R.; Blanchard, V. Acute-Phase Glycoprotein N-
Glycan Analysis Using a Flow-through Microvial Interface. Anal. Chem. Glycome of Ovarian Cancer Patients Analyzed by CE-LIF. Electro-
2014, 86, 6479−6486. phoresis 2016, 37, 1461−1467.
(104) Snyder, C. M.; Zhou, X.; Karty, J. A.; Fonslow, B. R.; Novotny, (122) Robajac, D.; Masnikosa, R.; Vanhooren, V.; Libert, C.;
M. V.; Jacobson, S. C. Capillary Electrophoresis−Mass Spectrometry Miković, Ž .; Nedić, O. The N-Glycan Profile of Placental Membrane
for Direct Structural Identification of Serum N-Glycans. J. Chromatogr. Glycoproteins Alters During Gestation and Aging. Mech. Ageing Dev.
A 2017, 1523, 127−139. 2014, 138, 1−9.

7884 DOI: 10.1021/acs.chemrev.7b00669

Chem. Rev. 2018, 118, 7867−7885
Chemical Reviews Review

(123) Konze, S. A.; Cajic, S.; Oberbeck, A.; Hennig, R.; Pich, A.; (140) Gatea, F.; et al. Chemical Constituents and Bioactive Potential
Rapp, E.; Buettner, F. F. R. Quantitative Assessment of Sialo- of Portulaca Pilosa L Vs. Portulaca Oleracea L. Med. Chem. Res. 2017,
Glycoproteins and N-Glycans During Cardiomyogenic Differentiation 26, 1516−1527.
of Human Induced Pluripotent Stem Cells. ChemBioChem 2017, 18, (141) Dominguez, M. A.; Jacksén, J.; Emmer, Å.; Centurión, M. E.
1317−1331. Capillary Electrophoresis Method for the Simultaneous Determination
(124) Zhang, L.; Lawson, K.; Yeung, B.; Wypych, J. Capillary Zone of Carbohydrates and Proline in Honey Samples. Microchem. J. 2016,
Electrophoresis Method for a Highly Glycosylated and Sialylated 129, 1−4.
Recombinant Protein: Development, Characterization and Application (142) Toutounji, M. R.; Van Leeuwen, M. P.; Oliver, J. D.; Shrestha,
for Process Development. Anal. Chem. 2015, 87, 470−476. A. K.; Castignolles, P.; Gaborieau, M. Quantification of Sugars in
(125) Gahoual, R.; Biacchi, M.; Chicher, J.; Kuhn, L.; Hammann, P.; Breakfast Cereals Using Capillary Electrophoresis. Carbohydr. Res.
Beck, A.; Leize-Wagner, E.; François, Y. N. Monoclonal Antibodies 2015, 408, 134−141.
Biosimilarity Assessment Using Transient Isotachophoresis Capillary (143) Navarro-Pascual-Ahuir, M.; Lerma-García, M. J.; Simó-Alfonso,
Zone Electrophoresis-Tandem Mass Spectrometry. mAbs 2014, 6, E. F.; Herrero-Martínez, J. M. Rapid Differentiation of Commercial
1464−1473. Juices and Blends by Using Sugar Profiles Obtained by Capillary Zone
(126) Redman, E. A.; Mellors, J. S.; Starkey, J. A.; Ramsey, J. M. Electrophoresis with Indirect UV Detection. J. Agric. Food Chem. 2015,
Characterization of Intact Antibody Drug Conjugate Variants Using 63, 2639−2646.
Microfluidic Capillary Electrophoresis−Mass Spectrometry. Anal. (144) Alinat, E.; Jemmali, S.; Delaunay, N.; Archer, X.; Gareil, P.
Chem. 2016, 88, 2220−2226. Analysis of Underivatized Cellodextrin Oligosaccharides by Capillary
(127) Redman, E. A.; Ramos-Payan, M.; Mellors, J. S.; Ramsey, J. M. Electrophoresis with Direct Photochemically Induced UV-Detection.
Analysis of Hemoglobin Glycation Using Microfluidic Ce-Ms: A Electrophoresis 2015, 36, 1555−1563.
Rapid, Mass Spectrometry Compatible Method for Assessing Diabetes (145) Moreno, D.; Berli, F.; Bottini, R.; Piccoli, P. N.; Silva, M. F.
Management. Anal. Chem. 2016, 88, 5324−5330. Grapevine Tissues and Phenology Differentially Affect Soluble
(128) Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, Carbohydrates Determination by Capillary Electrophoresis. Plant
K.; Bisio, A.; Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; et al. Physiol. Biochem. 2017, 118, 394−399.
Oversulfated Chondroitin Sulfate Is a Contaminant in Heparin (146) Francisco, K. J. M.; do Lago, C. L. A Capillary Electrophoresis
Associated with Adverse Clinical Events. Nat. Biotechnol. 2008, 26, System with Dual Capacitively Coupled Contactless Conductivity
669−675. Detection and Electrospray Ionization Tandem Mass Spectrometry.
(129) Liu, H.; Zhang, Z.; Linhardt, R. J. Lessons Learned from the Electrophoresis 2016, 37, 1718−1724.
Contamination of Heparin. Nat. Prod. Rep. 2009, 26, 313−321. (147) Mar, N. N.; Umemoto, T.; Abdulah, S. N. A.; Maziah, M.
(130) Sun, X.; Lin, L.; Liu, X.; Zhang, F.; Chi, L.; Xia, Q.; Linhardt, Chain Length Distribution of Amylopectin and Physicochemical
R. J. Capillary Electrophoresis−Mass Spectrometry for the Analysis of Properties of Starch in Myanmar Rice Cultivars. Int. J. Food Prop.
Heparin Oligosaccharides and Low Molecular Weight Heparin. Anal. 2015, 18, 1719−1730.
Chem. 2016, 88, 1937−1943. (148) Wu, A. C.; Li, E.; Gilbert, R. G. Exploring Extraction/
(131) Lin, L.; Liu, X.; Zhang, F.; Chi, L.; Amster, I. J.; Leach, F. E.; Dissolution Procedures for Analysis of Starch Chain-Length
Xia, Q.; Linhardt, R. J. Analysis of Heparin Oligosaccharides by Distributions. Carbohydr. Polym. 2014, 114, 36−42.
Capillary Electrophoresis−Negative-Ion Electrospray Ionization Mass (149) Bai, Y.; Wu, P.; Wang, K.; Li, C.; Li, E.; Gilbert, R. G. Effects of
Spectrometry. Anal. Bioanal. Chem. 2017, 409, 411−420. Pectin on Molecular Structural Changes in Starch During Digestion.
(132) Maccari, F.; Galeotti, F.; Zampini, L.; Padella, L.; Tomanin, R.; Food Hydrocolloids 2017, 69, 10−18.
Concolino, D.; Fiumara, A.; Galeazzi, T.; Coppa, G.; Gabrielli, O.; (150) Yu, W.; Tan, X.; Zou, W.; Hu, Z.; Fox, G. P.; Gidley, M. J.;
et al. Total and Single Species of Uronic Acid-Bearing Glycosamino- Gilbert, R. G. Relationships between Protein Content, Starch
glycans in Urine of Newborns of 2−3 days of Age for Early Diagnosis Molecular Structure and Grain Size in Barley. Carbohydr. Polym.
Application. Clin. Chim. Acta 2016, 463, 67−72. 2017, 155, 271−279.
(133) Ucakturk, E.; Cai, C.; Li, L.; Li, G.; Zhang, F.; Linhardt, R. J. (151) Ibrahim, Y. M.; Hamid, A. M.; Deng, L.; Garimella, S. V. B.;
Capillary Electrophoresis for Total Glycosaminoglycan Analysis. Anal. Webb, I. K.; Baker, E. S.; Smith, R. D. New Frontiers for Mass
Bioanal. Chem. 2014, 406, 4617−4626. Spectrometry Based Upon Structures for Lossless Ion Manipulations.
(134) Zhao, T.; Song, X.; Tan, X.; Xu, L.; Yu, M.; Wang, S.; Liu, X.; Analyst 2017, 142, 1010−1021.
Wang, F. Development of a Rapid Method for Simultaneous (152) Szigeti, M.; Guttman, A. Automated N-Glycosylation
Separation of Hyaluronic Acid, Chondroitin Sulfate, Dermatan Sulfate Sequencing of Biopharmaceuticals by Capillary Electrophoresis. Sci.
and Heparin by Capillary Electrophoresis. Carbohydr. Polym. 2016, Rep. 2017, 7, 11663.
141, 197−203. (153) Redman, E. A.; Batz, N. G.; Mellors, J. S.; Ramsey, J. M.
(135) Restaino, O. F.; di Lauro, I.; Di Nuzzo, R.; De Rosa, M.; Integrated Microfluidic Capillary Electrophoresis-Electrospray Ioniza-
Schiraldi, C. New Insight into Chondroitin and Heparosan-Like tion Devices with Online MS Detection for the Separation and
Characterization of Intact Monoclonal Antibody Variants. Anal. Chem.
Capsular Polysaccharide Synthesis by Profiling of the Nucleotide Sugar
2015, 87, 2264−2272.
Precursors. Biosci. Rep. 2017, 37 (1−11), BSR20160548.
(154) Szarka, M.; Guttman, A. Smartphone Cortex Controlled Real-
(136) Witos, J.; Samuelsson, J.; Cilpa-Karhu, G.; Metso, J.;
Time Image Processing and Reprocessing for Concentration
Jauhiainen, M.; Riekkola, M.-L. Partial Filling Affinity Capillary
Independent LED Induced Fluorescence Detection in Capillary
Electrophoresis Including Adsorption Energy Distribution Calcula-
Electrophoresis. Anal. Chem. 2017, 89, 10673−10678.
tions - Towards Reliable and Feasible Biomolecular Interaction
Studies. Analyst 2015, 140, 3175−3182.
(137) Liang, A.; Desai, U. R. In Glycosaminoglycans: Chemistry and
Biology; Balagurunathan, K., Nakato, H., Desai, U. R., Eds.; Humana
Press: New York, NY, 2015, 1229, 355−375.
(138) Oliveira, D. L.; et al. Natural Caprine Whey Oligosaccharides
Separated by Membrane Technology and Profile Evaluation by
Capillary Electrophoresis. Food Bioprocess Technol. 2014, 7, 915−920.
(139) Lu, Y.; Hu, Y.; Wang, T.; Yang, X.; Zhao, Y. Rapid
Determination and Quantitation of Compositional Carbohydrates to
Identify Honey by Capillary Zone Electrophoresis. CyTA–J. Food
2017, 15, 531−537.

7885 DOI: 10.1021/acs.chemrev.7b00669

Chem. Rev. 2018, 118, 7867−7885