Structure analysis of N-glycoproteins.

Abstract

General mass spectrometry-based strategies for analysis of N-glycosylated peptides are described. The well-established method utilizes Peptide-N-glycosidase F (PNGase F) for in-gel or in-solution release of N-linked glycans from the polypeptide chains (along with the conversion of the formerly N-glycosylated Asn to Asp), thus allowing separate analysis of glycan moieties and deglycosylated peptides. However, no assignment of individual glycans to a glycosylation site can be realized. Intact glycopeptides (i.e., proteolytic mixtures in which the glycan chains stay attached at their original glycosylation sites) can be analyzed either by a direct infusion or with HPLC separation prior to MALDI or ESI mass spectrometric analysis to provide both information on the glycan structure and glycosylation site in the same experiment. Several different strategies for efficient in-solution digestion of glycoproteins are described, such as proteolytic digestion in the electrospray capillary and simultaneous analysis of the resulting (glyco)peptides.