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


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
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Early online date19 Apr 2021
Publication statusE-pub ahead of print - 19 Apr 2021


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

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