Use of a lectin affinity selector in the search for unusual glycosylation in proteomics

The purpose of the work described in this paper was to develop a new approach to the identification of glycoprotein with particular types of glycosylation. The paper demonstrates N-glycosylation sites in a glycoproteins can be identified by (1) proteolysis with trypsin, (2) lectin affinity selection, (3) enzymatic deglycosylation with peptide-N-glycosidase F (PNGase F) in buffer containing 95% H(2)(18)O, which generates deglycosylated peptide pairs separated by 2 or 4 amu, (4) reversed-phase separation of the peptide mixture and MALDI mass analysis, (5) MS-MS sequencing of the ion pairs, and (6) identification of the parent protein through a database search. This process has been tested on the selection of glycopeptides from lactoferrin and mammaglobin, and the identification of the ion pairs of fetuin glycopeptides. Glycosylation sites were identified through PNGase hydrolysis in H(2)(18)O. During the process of hydrolyzing the conjugate, Asn is converted to an aspartate residue with the incorporation of (18)O. However, PNGase F was observed to incorporate two (18)O into the beta-carboxyl groups of the Asp residue. This suggests that the hydrolysis is at least partially reversible.