Partial vapor-phase hydrolysis of peptide bonds: A method for mass spectrometric determination of O-glycosylated sites in glycopeptides

In this study we present a method for determination of O-glycosylation site s in glycopeptides, based on partial vapor-phase acid hydrolysis in combina tion with mass spectrometric analysis. Pentafluoropropionic acid and hydroc hloric acid were used for the hydrolysis of glycosylated peptides. The reac tion conditions were optimized for efficient polypeptide backbone cleavages with minimal cleavage of glycosidic bonds. The glycosylated residues were identified hg mass spectrometric analysis of the hydrolytic cleavage produc ts. Although glycosidic bonds are partially cleaved under acid hydrolysis, the resulting mass spectra allowed unambiguous determination of the glycosy lation sites. Examples are shown with mannosyl- and mucin-type glycopeptide s. Performing the hydrolysis in vapor eliminates the risk for contamination of the sample with impurities fi om the reagents, thus allowing analysis o f the reaction products without further purification both by matrix-assiste d laser desorption/ionization and electrospray ionization mass spectrometry . (C) 1999 Academic Press.