Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments

In-gel digestion of densely O-glycosylated proteins, an essential step in p roteome analysis, is often hampered by steric hindrance of the proteases, T o overcome this technical problem a simple and convenient method has been d eveloped, which combines several advantages: (1) Approximately 70% of the o ligosaccharides are cleaved without significant protein hydrolysis at the o ptimal reaction conditions of 70% ethylamine, and quantitative cleavage is achieved with 40% methylamine, at 50 degreesC. (2) To the unsaturated deriv atives of Ser and Thr the alkylamine is added as a label of previous O-glyc osylation sites. (3) The alkylaminylated protein is effectively cleaved by proteolysis. (4) The modified peptides are identified by MALDI mass spectro metry under consideration of incremental mass increases. (5) The alkylamine label is stable under MALDI post-source-decay analysis as well as in colli sion-induced dissociation experiments allowing sequencing and peptide local ization of O-glycosylation:sites. Applicability of the method is evaluated with a series of synthetic glycopeptides, the densely O-glycosylated human glycophorin A, and with the mucin MUC1 from human milk fat globule membrane s. (C) 2001 Academic Press.