Capillary electrophoresis–mass spectrometry for direct structural identification of serum N-glycans
Open accessAbstract
Through direct coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with a sheathless interface, we have identified 77 potential N-glycan structures derived from human serum. We confirmed the presence of N-glycans previously identified by indirect methods, e.g., electrophoretic mobility standards, obtained 31 new N-glycan structures not identified in our prior work, differentiated co-migrating structures, and determined specific linkages on isomers featuring sialic acids. Serum N-glycans were cleaved from proteins, neutralized via methylamidation, and labeled with the fluorescent tag 8-aminopyrene-1,3,6-trisulfonic acid, which renders the glycan fluorescent and provides a -3 charge for electrophoresis and negative-mode MS detection. The neutralization reaction also stabilizes the labile sialic acids. In addition to methylamidation, native charges from sialic acids were neutralized through reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium to amidate α2,6-linked sialic acids in the presence of ammonium chloride and form lactones with α2,3-linked sialic acids. This neutralization effectively labels each type of sialic acid with a unique mass to determine specific linkages on sialylated N-glycans. For both neutralization schemes, we compared the results from microchip electrophoresis and CE.
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The sheathless interface has been widely used for ultrasensitive proteomics analysis, although it is limited by potential pore blocking.
“The sheathless interface has been widely used in proteomics research to provide ultrasensitive analysis56–58, although it is limited by potential blocking of the pores.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [57]
CZE separation has frequently been used to analyze glycosylated peptides and glycans, and CZE via TDMS has been used for glycosylation characterization.
“CZE separation has frequently been used to analyze glycosylated peptides and glycans49,57,80,81 and CZE separation via TDMS was used for glycosylation characterization82.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [57]
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Advances in mass spectrometry‐based glycomics
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MS‐based glycomics and glycoproteomics methods enabling isomeric characterization
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Capillary zone electrophoresis-mass spectrometry for bottom-up proteomics
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Mass spectrometry for structural elucidation and sequencing of carbohydrates
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Recent Advances in Mass Spectrometry-Based Structural Elucidation Techniques
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NEGATIVE ION MASS SPECTROMETRY FOR THE ANALYSIS OF N‐LINKED GLYCANS
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Highly-sensitive label-free deep profiling of N-glycans released from biomedically-relevant samples
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Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis
Sârbu, M., & Zamfir, A. D. (2018) · Electrophoresis · doi:10.1002/elps.201700461 · Cited by 26 · see citing works
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Expanding the Reaction Space of Linkage-Specific Sialic Acid Derivatization
Pongrácz, T., Wuhrer, M., & de Haan, N. (2019) · Molecules · doi:10.3390/molecules24193617 · Cited by 24 · see citing works
Labeling strategies for analysis of oligosaccharides and glycans by capillary electrophoresis
Smolkova, D., Čmelík, R., & Lavická, J. (2023) · TrAC Trends in Analytical Chemistry · doi:10.1016/j.trac.2023.117068 · Cited by 24 · see citing works
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Capillary (Gel) Electrophoresis-Based Methods for Immunoglobulin (G) Glycosylation Analysis
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MS-based glycomics: An analytical tool to assess nervous system diseases
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“The selection of CE types were based on different analytical purposes: CZE, the most popular type, provides ultrahigh resolution (a million plates) and very low sample injection volume (nanoliters); MCE, an alternative to CZE, provides faster speed, higher resolution, lower sample volumes, and multiple functions on a…”
Characterization of glycan isomers using magnetic carbon nanoparticles as a MALDI co-matrix
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State-of-the-Art Glycomics Technologies in Glycobiotechnology
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Emerging technologies for single-cell glycomics
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A survey of electrokinetically‐driven microfluidics for cancer cells manipulation
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Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics
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Recent applications of the derivatization techniques in capillary electrophoresis
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LC-MS/MS in glycomics and glycoproteomics analyses
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“The serum N-glycan numbers roughly agree with those seen in our previous investigations [8, 23, 40] and those reported elsewhere [42].”
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Hitting the sweet spot with capillary electrophoresis: advances in N-glycomics and glycoproteomics
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Fractionation and characterization of sialyl linkage isomers of serum N‐glycans by CE–MS
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Self-citationSARS-CoV-2 Attacks in the Brain: Focus on the Sialome
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Multilevel Characterization of Antibody-Ligand Conjugates by CESI-MS
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Self-citationMass Spectrometric Analysis of Protein Glycosylation
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Advances in mass spectrometry-based strategies for analyzing sialic acid linkage isomers
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CE–MS Approaches for Glyco(proteo)mic Analysis
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Advances in MS-based glycomics for biomedical research
Solomon, J., Sanni, A., Purba, W. T., Oluokun, O., Sahioun, S., Fowowe, M., Oluokun, A., Hussein, A., & Mechref, Y. S. (2025) · Analytical Methods · doi:10.1039/d5ay01169h · Cited by 1 · see citing works
- Authors
- Snyder, C. M., Zhou, X., Karty, J. A., Fonslow, B. R., Novotny, M. V., & Jacobson, S. C.
- Affiliations
- Indiana University Bloomington (US)
- Corresponding author
- Jacobson, Stephen C.
- Venue
- Journal of Chromatography A
- Publisher
- Elsevier BV
- Published
- 2017-11
- Volume
- 1523
- Pages
- 127-139
- ISSN
- 0021-9673 (ISSN-L)
- PMC
- PMC5691604
- OpenAlex
- W2750924492
- Cited by
- 58
- References
- 67
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 3 · 2023: 4 · 2022: 8 · 2021: 7 · 2020: 11 · 2019: 10 · 2018: 11
- FWCI
- 3.58
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Journal of Chromatography A (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- IUScholarWorks (Indiana University) (submittedVersion)
- Funders
- Indiana University Nanoscale Characterization Facility for use of its instruments (NIH R01 GM106084)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Keywords
- N-glycans, serum glycoproteins, α2,6-linked sialic acid, α2,3-linked sialic acid, glycan isomers, microchip electrophoresis
- MeSH terms
- Blood Chemical Analysis, Humans, Isomerism, Polysaccharides, Pyrenes, Mass Spectrometry, Electrophoresis, Capillary, N-Acetylneuraminic Acid
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