Identification of protein-bound carbohydrates by mass spectrometry

Harvey, David J.

doi:10.1002/1615-9861(200102)1:2<311::aid-prot311>3.0.co;2-j

Cited by 169 publications

(103 citation statements)

References 107 publications

(149 reference statements)

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“…Assignment of glycan monosaccharide composition could be based on the observed precursor mass alone because of the restricted combinations of monosaccharide residues of N-glycans. 52 For each glycan monosaccharide composition there are several possible isomeric structures and different types of glycosidic linkages that cannot be identified by single stage CE-MS analysis. Differences in electrophoretic mobilities, resulting in differential migration time for glycans, can be useful to predict the structure of certain glycans.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

et al. 2014

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A flow-through microvial is used to interface capillary electrophoresis and mass spectrometry (CE-MS) to develop a method for simultaneous profiling both neutral and sialylated glycans without derivatization or labeling. The CE separation was performed at near-zero electroosmotic flow in a capillary with neutral, hydrophilic coating, using 50 mM ammonium acetate in 20% methanol (pH 3.1) as the background electrolyte. The method was optimized with reversed CE polarity and negative ion ESI-MS. Enzymatically released N-glycans from human immunoglobulin G (IgG) were used as the test sample. The approach was also used to study the more complex N-glycans from recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary (CHO) cells. Glycoscreening of rHuEPO was performed using a triple quadrupole MS and an ultrahigh resolution TOF-MS. The high sensitivity and high mass accuracy of the TOF-MS revealed the presence of more than 70 glycans. Three mono-and di-sialylated tetra-antennary N-glycans and one mono-sialylated tri-antennary N-glycan of rHuEPO are reported for the first time. Further glycan heterogeneity was identified of the highly sialylated N-glycans of rHuEPO by extensive acetylation, Neu5Ac/Neu5Gc variation and the presence of N-acetyl-lactosamine repeats. For comparative purposes, porous graphitic carbon-based LC-MS/MS was also used to glycoprofile rHuEPO. This work demonstrates the potential of CE-MS to provide a comprehensive glycosylation profile with detailed features of the secondary glycan modifications. The CE-MS based method eliminates the need to label the N-glycans, as well as the requirement to desialylate before analysis, and could complement other established techniques for glycan characterization of therapeutic glycoproteins.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

et al. 2014

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“…The knowledge obtained from fragmentation of small oligosaccharides was used for interpreting spectra of larger and more complex structures. In Figure 3 it is shown that the branching pattern can be determined for four different spectra of Core 4 hexasaccharides with the same composition dHex 1 …”

Section: Fragmentation Of [M ϫ H] ϫ Ions Of Neutral Mucin Oligosacchamentioning

confidence: 99%

“…This increase in interest has worked hand in hand with the development of more sensitive and specific analytical techniques in this field. Mass spectrometry has been the detection and characterization tool most frequently used in recent years for analysis of glycoproteins, glycopeptides, glycolipids, or free oligosaccharides (some reviews in [1][2][3][4][5]). In principle, mass spectrometry suffers from the fact that the monomeric components (the monosaccharides) of oligosaccharide chains consist of isomeric building blocks, and that linkage configuration and position are difficult to obtain with the technique.…”

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confidence: 99%

Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSⁿ.

Karlsson¹,

Schulz²,

Packer³

2004

J. Am. Soc. Mass Spectrom.

127

133

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Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were separated and detected by negative ion LC-MS and LC-MS 2 . The resolution of the chromatography and the information obtained from collision induced dissociation of detected [M Ϫ H] Ϫ ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nonasaccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Z i and Y i ), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAc␤1-3GalNAcol or Gal/GlcNAc␤1-3(GlcNAc␤1-6)GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and A i type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution ( 0,2 A i Ϫ H 2 O cleavage) and disubstitution of C-3 and C-4 (Z i /Z i cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Gal␤1-3/ 4GlcNAc␤1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, negative ion LC-MS 2 was able to generate extensive data for understanding the overall glycosylation pattern of a sample, especially when only a limited amount of material is available. I dentification and understanding of biological processes involving glycoconjugates is a scientific area of increasing interest. This increase in interest has worked hand in hand with the development of more sensitive and specific analytical techniques in this field. Mass spectrometry has been the detection and characterization tool most frequently used in recent years for analysis of glycoproteins, glycopeptides, glycolipids, or free oligosaccharides (some reviews in [1][2][3][4][5]). In principle, mass spectrometry suffers from the fact that the monomeric components (the monosaccharides) of oligosaccharide chains consist of isomeric building blocks, and that linkage configuration and position are difficult to obtain with the technique. Even so, researchers have applied mass spectrometric techniques to answer biological questions (some examples in [6 -9]).Characterization of released or free oligosaccharides by mass spectrometry using electrospray ionization in negative mode has recently been shown to give both sequence and linkage information [10,11]. Negative ion mode has been shown to be applicable to both negatively charged oligosaccharides and for neutral oligosaccharides [12][13][14], and offers high sensitivity of detection without requiring derivatization or adduct formation to aid ionization. Electrospray is also easily coupled to with high resolution liquid chromatography, an important factor in resolving the degeneracy of structural isomers found i...

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“…Glycosylation is one of the major and most complicated forms of post-translational modifications that plays essential roles in the activity and effector function of monoclonal antibodies [3] demanding improved glycoanalytical methods. Frequently 3/13 used techniques for carbohydrate analysis are mass spectrometry (MS) [4], liquid chromatography (LC) [5,6], capillary electrophoresis (CE) [7][8][9], some combination of them [10][11][12] and if necessary, nuclear magnetic resonance spectroscopy (NMR) [13,14].…”

Section: Introductionmentioning

confidence: 99%

Evaporative fluorophore labeling of carbohydrates via reductive amination

Reider

Szigeti

Guttman

2018

Talanta

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As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol with the great advantage of continuously concentrating the reagents during the tagging reaction, therefore accommodating to reach the optimal reagent concentrations for a wide range of glycan structures in a complex mixture. The optimized conditions of the evaporative labeling process minimized sialylation loss, otherwise representing a major issue in reductive amination based carbohydrate tagging. In addition, complete and uniform dispersion of dry samples was obtained by supplementing the low volume labeling mixtures (several microliters) with the addition of extra solvent (e.g., THF).Evaporative labeling is an automation-friendly glycan labeling method, suitable for standard open 96 well plate format operation. Graphical abstract 2/13

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Identification of protein-bound carbohydrates by mass spectrometry

Cited by 169 publications

References 107 publications

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSⁿ.

Evaporative fluorophore labeling of carbohydrates via reductive amination

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Identification of protein-bound carbohydrates by mass spectrometry

Cited by 169 publications

References 107 publications

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn.

Evaporative fluorophore labeling of carbohydrates via reductive amination

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Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSⁿ.