Recently, proteolytic 18O labeling has been demonstrated as a promising strategy for comparative proteomic studies (Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-42). In this approach, protein mixtures are digested in parallel in H216O and H218O and the ratios of isotopically distinct peptide products are measured by mass spectrometry. In the initial report from this laboratory, trypsin was shown to catalyze incorporation of two 18O atoms into the carboxyl terminus of each new peptide formed by cleavage of the adenovirus proteome. In the present study, a second enzyme, endoprotease Glu-C, is evaluated as an agent for cleavage and labeling. Proteolytic 18O labeling by Glu-C is shown to occur readily with phosphorylated and glycosylated proteins and with cysteinealkylated and disulfide-linked proteins. A sequential double-labeling strategy is used to characterize N-linked glycopeptides. Labeled and unlabeled peptide pairs are found to coelute chromatographically, and measurements of isotope ratios by nanospray and capillary LC-MS are found to be accurate and precise.