Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses

Beatriz Calle, Ganka Bineva-Todd, Andrea Marchesi, Helen Flynn, Mattia Ghirardello, Omur Y Tastan, Chloe Roustan, Junwon Choi, M Carmen Galan, Benjamin Schumann*, Stacy A Malaker*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

16 Citations (Scopus)
46 Downloads (Pure)


Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogues that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analogue glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal reporter reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

Original languageEnglish
Pages (from-to)2366-2375
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Issue number9
Early online date19 Apr 2021
Publication statusPublished - 1 Sept 2021

Bibliographical note

Funding Information:
The authors thank all of our past and current colleagues for their help with the work featured here. We are especially appreciative of Carolyn R. Bertozzi, Jeffrey Shabanowitz, and Donald F. Hunt for their mentorship, guidance, and support. We are grateful to Nicola O’Reilly and Dhira Joshi of the Francis Crick Institute Peptide Chemistry Science Technology Platform, to Paul C. Driscoll, Nathalie Legrave, and James MacRae of the Francis Crick Institute Metabolomics Science Technology Platform, and to Svend Kjaer and Phil Walker of the Francis Crick Institute Structural Biology Science Technology Platform. S.A.M. was supported by a Yale Science Development Fund. B.C. was supported by a Crick-HEI studentship funded by the Department of Chemistry at Imperial College London and the Francis Crick Institute. G.B.-T., O.Y.T. and B.S were supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001749), the UK Medical Research Council (FC001749), and the Wellcome Trust (FC001749). A.M. was supported by a Wellcome Trust Grant (218304/Z/19/Z). M.C.G. and M.G. were supported by the European Research Council (ERC-COG 648239) and the UK Biotechnology and Biological Sciences Research Council (BB/M028976/1). J.C. was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (2020R1C1C1005563). This research was funded in whole, or in part, by the Wellcome Trust (FC001749 and 218304/Z/19/Z). For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

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  • glycopeptide
  • glycoproteomics
  • click chemistry
  • bioorthogonal mucin
  • ETD


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