Evaluation of non‐reductive β‐elimination/Michael addition for glycosylation site determination in mucin‐like O‐glycopeptides

Abstract

Investigation of site-specific protein O-glycosylation remains a formidable task in post-translational modification-centred proteomics. In particular, the determination of O-glycosylated amino acids in mucin-like glycopeptides lags far behind the techniques for phosphorylation site and N-glycosylation site identification, for which well-established enrichment techniques are available. The present work investigated β-elimination of mucin-like O-glycopeptides with a mild alkylamine base and concomitant Michael-type addition using 2-mercaptoethanol as nucleophile applied to synthetic GalNAcylated O-glycopeptides as well as exoglycosidase-treated endogenous peptides isolated from human blood plasma. This strategy permits O-glycosylated sites to be unambiguously localized, even in multiple-glycosylated peptides. Peptides covalently modified with the glycan surrogate exhibit excellent backbone fragmentation in MS/MS due to their stability during CID.

High-sensitivity Analytical Approaches for the Structural Characterization of Glycoproteins

“…352 In a similar study, β -mercaptoethanol was used as the nucleophile when methylamine was used to induce β -elimination. 353 Similarly to the study using peptides modified with ethanethiol, 352 the β -mercaptoethanol tag appeared to be stable under MS 2 conditions. In an interesting modification, dithiothreitol (DTT) was used as a nucleophile to indicate the site of O-glycosylation.…”

Structural Biology of Glycoproteins

“…However, Halfinger et al have developed a robust protocol using partial digestion with exoglycosidase and -elimination using a mild alkylamine base followed by Michael addition and analysis using collision induced dissociation (CID) MS/MS [78].…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…Halfinger et al (2011) have developed a modified protocol involving exoglycosidase digestion and tryptic digestion to produce GalNAc-substituted peptides, removal of the GalNAc by b-elimination with dimethylamine, and Michael addition of 2-mercaptoethanol accompanied with a small amount of dimethylamine addition. The resulting peptides exhibited mass tags of 60 and 27 U, respectively, allowing the glycosylation sites to be determined, even with multiple glycosylated peptides.…”