Isomeric Separation of N-Glycopeptides Derived from Glycoproteins by Porous Graphitic Carbon (PGC) LC-MS/MS.

Protein glycosylation is involved in many biological processes and physiological functions. Despite the recent advances in LC-MS/MS methodologies, the profiling of site-specific glycosylation is one of the major analytical challenges of glycoprotein analysis. Herein, we first reported the separation of glycopeptide isomers on porous graphitic carbon (PGC) LC was significantly improved by elevating the separation temperature under basic mobile phases. These findings permitted the isomeric separation of glycopeptides resulting from highly specific enzymatic digestions. The selectivity for different glycan types were studied using bovine fetuin, asialofetuin, IgG, ribonuclease B glycoproteins by PGC-LCMS. Comprehensive structural isomeric separation of glycopeptides was observed by high resolution MS and MS/MS. The specific structures of the glycopeptide isomers were identified and confirmed through exoglycosidase digestions. The glycosylation analysis of human alpha-1 acid glycoprotein revealed the potential use of PGC-LCMS for complex glycoprotein analysis for biomarker discovery. This newly developed separation method was shown as a reliable analytical method providing complete isomeric separation of site-specific glycoprotein analysis.