Site-Specific Glycan Heterogeneity Characterization by Hydrophilic Interaction Liquid Chromatography Solid-Phase Extraction, Reversed-Phase Liquid Chromatography Fractionation, and Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry
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Reversed-phase chromatographic separation of glycopeptides tends to be dominated by the peptide composition. In contrast, capillary zone electrophoresis separation of glycopeptides is particularly sensitive to the sialic acid composition of the glycan. In this paper, we combine the two techniques to achieve superior N-glycopeptide analysis. Glycopeptides were first isolated from a tryptic digest using hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction. The glycopeptides were separated using reversed-phase ultra high-performance liquid chromatography (UHPLC) to generate four fractions corresponding to different peptide backbones. Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) was used to analyze the fractions. We applied this method for the analysis of alpha-1-acid glycoprotein (AGP). A total of 268 site-specific N-glycopeptides were detected, representing eight different glycosylation sites from two isomers of AGP. Glycans included tetra-sialic acids with multi N-acetyllactosamine (LacNAc) repeats and unusual pentasialylated terminal sialic acids. Reversed-phase UHPLC coupled with CZE generated ∼35% more N-glycopeptides than direct reversed-phase UHPLC-ESI-MS/MS analysis and ∼70% more N-glycopeptides than direct CZE-ESI-MS/MS analysis. This approach is a promising tool for global, site-specific glycosylation analysis of highly heterogeneous glycoproteins with mass-limited samples.
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Qu and co-workers reported an LC-CZE strategy for site-specific glycan heterogeneity in which CZE analysis of reversed-phase LC glycopeptide fractions reduced sample complexity and enabled glycan structural elucidation.
“Qu and co-workers reported a LC-CZE strategy for the characterization of site-specific glycan heterogeneity where the use of CZE for analysis of reversed-phase LC glycopeptide fractions reduced the complexity of the sample and enabled glycan structural elucidation88.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [88]
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- Authors
- Qu, Y., Sun, L., Zhang, Z., & Dovichi, N. J.
- Affiliations
- University of Notre Dame (US)
- Michigan State University (US)
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2018-01-03
- Volume
- 90
- Issue
- 2
- Pages
- 1223-1233
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC5771954
- OpenAlex
- W2772801344
- Cited by
- 40
- References
- 27
- Citations by year
- 2026: 3 · 2024: 2 · 2023: 5 · 2022: 5 · 2021: 8 · 2020: 6 · 2019: 6 · 2018: 5
- FWCI
- 1.90
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of General Medical Sciences (R01 GM096767)
- 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 › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Keywords
- site-specific glycosylation, N-glycopeptides, glycan heterogeneity, alpha-1-acid glycoprotein, sialic acid, glycopeptide isolation, capillary zone electrophoresis, tandem mass spectrometry, glycoprotein analysis
- MeSH terms
- Animals, Cattle, Chromatography, High Pressure Liquid, Glycopeptides, Glycoproteins, Glycosylation, Humans, Orosomucoid, Polysaccharides, Electrophoresis, Capillary, Spectrometry, Mass, Electrospray Ionization, Solid Phase Extraction, Tandem Mass Spectrometry, Chromatography, Reverse-Phase, Hydrophobic and Hydrophilic Interactions
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