Capillary electrophoresis-electrospray mass spectrometry for the characterization of high-mannose-type N-glycosylation and differential oxidation in glycoproteins by charge reversal and protease/glycosidase digestion

The characterization of high-mannose-type N-glycosylation by capillary electrophoresis-electrospray mass spectrometry (CE-ESI MS) was described. In addition to the use of a cationic noncovalent capillary coating, strong acidic buffer, and charge reversal to increase the glycoform resolving power, N-glycosidase F (PNGase F) combined with a basic protease and a-mannosidase combined with an acidic protease were used to analyze the high, mannose-type N-glycosylation in ribonuclease B (RNase B) and in a novel C-type lectin from the venom of Trimeresurus stejnegeri (TSL). The structures of oligosaccharide, glycosylation sites, and glycoform distributions were determined simultaneously, and the differential oxidation of Met residues in glycopeptides obtained from TSL protease digestion was also characterized successfully by CE-MS/MS. The results showed that the oligosaccharide attached to RNase B has a structure of GlcNAc(2)Man(5 similar to9), and that attached to TSL has a structure of GlcNAc(2)Man(5 similar to8), The glycoform distributions in these glycoproteins are quite different, with the GlcNAc(2)Man(5) type predominant in RNase B, and the GlcNAc(2)Man(8) type, in TSL. This method may be useful not only for the characterization of glycosylation sites and glycan structures, but also for the determination of the relative abundance of individual glycoforms.