Capillary zone electrophoresis and MALDI-mass spectrometry for the monitoring of in vitro O-glycosylation of a threonine/serine-rich MUC5AC hexadecapeptide

The in vitro N-acerylgalactosaminylation by human gastric UDP-GalNAc: polyp eptide N-acetylgalactosaminyltransferases was assessed using the peptide mo tif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and th e given glycopeptide species were well separated by capillary zone electrop horesis. Moreover, the degree of glycosylation (number of monosaccharide O- linked attachments) could be determined by MALDI-mass spectrometry without prior separation. Using different incubation times, we evidenced the accumu lation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processin g. This information was completed by experimental data showing that N-acety lgalactosaminylated octapeptides (the peptide backbones: of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactos aminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-gl ycosylation process. (C) 1999 Elsevier Science B.V. All rights reserved.