title: 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.
authors.affiliation: Division of Clinical Biochemistry and Protein Micro-Analysis Facility, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Halfinger
authors.forename: Bettina
authors.initials: B
authors.lastname: Sarg
authors.forename: Herbert H
authors.initials: HH
authors.lastname: Lindner
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D006020:Glycopeptides / Q000097:blood* / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008623:Mercaptoethanol / Q000737:chemistry; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D001798:Blood Proteins; D006020:Glycopeptides; D009077:Mucins; D008623:Mercaptoethanol