Sensitive and fast characterization of site-specific protein glycosylation with capillary electrophoresis coupled to mass spectrometry
Open accessAbstract
Glycoproteomic analysis requires efficient separation and sensitive detection to enable the comprehensive characterization of glycan heterogeneity. Here, we report the use of capillary zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) with an electrokinetically-pumped nanospray interface for the study of protein glycosylation microheterogeneity. A fast separation was developed that resolved intact glycopeptides generated from standard proteins within ~9min. Differentially terminal-galactosylated and sialylated species with the same glycosylation sites were well resolved. The concentration detection limits for CZE were three times higher than for nanoLC methods; however, a 200-fold smaller injection volume was used in CZE, which reflects the use of an extremely efficient electrospray interface in our CZE-ESI-MS setup. The resulting glycopeptide mass detection limit was two orders of magnitude superior to a nanoLC method. We also observed a 1.5% and 7% average relative standard deviation in peak migration time and glycopeptide relative abundance, and a four order of magnitude linear dynamic range in signal intensity. With CZE-ESI-MS, 40 haptoglobin glycopeptides were identified from roughly 40 fmol of digest.
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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 [49]
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MethodRecent developments in capillary and microchip electroseparations of peptides (2017–mid 2019)
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- Authors
- Qu, Y., Sun, L., Zhu, G., Zhang, Z., Peuchen, E. H., & Dovichi, N. J.
- Affiliations
- University of Notre Dame (US)
- Venue
- Talanta
- Publisher
- Elsevier BV
- Published
- 2018-03
- Volume
- 179
- Pages
- 22-27
- ISSN
- 0039-9140 (ISSN-L)
- PMC
- PMC5763510
- OpenAlex
- W2766266487
- Cited by
- 30
- References
- 23
- Citations by year
- 2022: 3 · 2021: 9 · 2020: 7 · 2019: 8 · 2018: 3
- FWCI
- 2.01
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (am, 244 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Talanta (acceptedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (R01GM096767)
- National Science Foundation (2015-2018)
- 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
- protein glycosylation, glycopeptide separation, capillary zone electrophoresis, glycan heterogeneity, sialylation, haptoglobin, detection limit, glycoproteomics
- MeSH terms
- Acetylglucosamine, Amino Acid Sequence, Carbohydrate Sequence, Fucose, Galactose, Glycopeptides, Glycosylation, Haptoglobins, Humans, Mannose, Protein Processing, Post-Translational, Electrophoresis, Capillary, N-Acetylneuraminic Acid, Spectrometry, Mass, Electrospray Ionization, Limit of Detection
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