Abstract
Glycosylation is an essential protein modification occurring on the majority of extracellular human proteins, with mass spectrometry (MS) being an indispensable tool for its analysis, that not only determines glycan compositions, but also the position of the glycan at specific sites via glycoproteomics. However, glycans are complex branching structures with monosaccharides interconnected in a variety of biologically relevant linkages, isomeric properties that are invisible when the readout is mass alone. Here, we developed an LC-MS/MS-based workflow for determining glycopeptide isomer ratios. Making use of isomerically defined glyco(peptide) standards, we observed marked differences in fragmentation behavior between isomer pairs when subjected to collision energy gradients, specifically in terms of the galactosylation/sialylation branching and linkage. These behaviors were developed into component variables that allowed for relative quantification of isomerism within mixtures. Importantly, at least for small peptides, the isomer quantification appeared to be largely independent from the peptide portion of the conjugate, allowing a broad application of the method.
Original language | English |
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Pages (from-to) | 9605-9614 |
Number of pages | 10 |
Journal | Analytical Chemistry |
Volume | 95 |
Issue number | 25 |
DOIs | |
Publication status | Published - 27 Jun 2023 |
Bibliographical note
Publisher Copyright:© 2023 The Authors. Published by American Chemical Society.
Funding
We would like to thank Roche Diagnostics for kindly providing Pronase and tastuzumab, Dario Cramer for helpful initial digestion experiments, Hillary Hoppenbrouwers for her assistance with setting up cotton-HILIC-based SGP extraction from egg yolk powder, and Gerlof Bosman for his efforts on glycopeptide synthesis. Furthermore, we would like to thank Javier Sastre Toraño for providing the isomerically defined glycosylated asparagine standards. This project was funded by the Dutch Research Council (NWO) Project VI.Veni.192.058, awarded to K.R.R. G.-J.P.H.B. acknowledges the ERC Advanced Grant 101020769.
Funders | Funder number |
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Roche | |
European Research Council | 101020769 |
Nederlandse Organisatie voor Wetenschappelijk Onderzoek | VI.Veni.192.058 |