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Rapid Commun Mass Spectrom. 2002;16(1):27-34.

Identification of substituted sites on MUC5AC mucin motif peptides after enzymatic O-glycosylation combining beta-elimination and fixed-charge derivatization.

Author information

1
Laboratoire de Chimie Biologique, UMR 8576 CNRS, Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cédex, France.

Abstract

A strategy for determination of O-glycosylation site(s) in glycopeptides has been developed using model compounds obtained by enzymatic glycosylation (by human GaNTase-T2 isoform) on peptides derived from the human MUC5AC mucin tandem repeat motif. The beta-elimination-addition reaction (using dimethylamine and concomitantly ethanethiol) on the formerly glycosylated sites through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. After N-terminal derivatization by a phosphonium group, peptide sequencing was then carried out by nanospray tandem mass spectrometry experiments. The highly predictable fragmentation pathways of these fixed-charge phosphonium derivatives enable straightforward recognition of glycosylation site(s) based on the mass increment of +44 Da for originally glycosylated threonine compared to the mass of fragments containing nonglycosylated residues.

PMID:
11754244
DOI:
10.1002/rcm.532
[Indexed for MEDLINE]

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