Identification of substituted sites on MUC5AC mucin motif peptides after enzymatic O-glycosylation combining beta-elimination and fixed-charge derivatization.
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.
Czeszak
X
X
Laboratoire de Chimie Biologique, UMR 8576 CNRS, Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cédex, France.
Ricart
G
G
Tetaert
D
D
Michalski
J C
JC
Lemoine
J
J
D000595:Amino Acid Sequence; D002384:Catalysis; D004123:Dimethylamines; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D020080:Tandem Repeat Sequences
D004123:Dimethylamines; D006020:Glycopeptides; D009077:Mucins; C034516:dimethylamine