Analysis by electrospray mass spectrometry of glycopeptides from the in vitro O-glycosylation reaction using human mucin motif peptide.

A mucin-motif peptide in the one-letter code T T T P S P P M T T P I T P P A, representative of the human intestinal mucin tandem repeat sequence (MUC2), containing several threonine residues in clusters, was used as an acceptor substrate to investigate the effect of peptide structure on the activity of crude preparation of human gastric UDP-GalNAc:polypeptide N-acetyl galactosaminyltransferases. High-performance liquid chromatography was performed to separate the different products of the in vitro O-glycosylated reaction. The electrospray mass spectrometry was used to identify the different masses (m/z) of these products. Although the m/z of glycopeptide(s) could be higher than the detection limits of the spectrometer, an accurate study of the doubly charged ions allowed us to demonstrate the linkage of more than two sugars. Hence, the peptide MUC2 will accept at least four carbohydrate residues but the exact substituted positions should be confirmed by further sequence determination.