IMPROVED CAPILLARY ELECTROPHORETIC SEPARATION OF GLYCOSYLATED OLIGOPEPTIDES THROUGH ADDITION OF POLY(VINYL ALCOHOL), AND ANALYSIS BY ELECTROSPRAY MASS-SPECTROMETRY

A method for the analysis of O-glycosylation of peptides has been deve loped, combining capillary electrophoretic (CE) separation and electro spray ionization mass spectrometry. Synthetic peptides with apomucin ' tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the pept ides with human gastric microsomal homogenates containing N-acetylgala ctosamine transferase activity in the presence of uridyl diphosphate N -acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solve nt, resulting in a greatly improved separation of the up to five diffe rent glycoforms of peptides with lengths of 8, 16 or 23 amino acids, a nd the unglycosylated peptides, After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glyco sylation pattern was shown to depend on the amino acid sequence of the peptides. (C) 1998 Elsevier Science B.V.