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

Rui Zhu, Yifan Huang, Jingfu Zhao, Jieqiang Zhong, Yehia Mechref

Research output: Contribution to journalArticlepeer-review

34 Scopus citations


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 report that 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 was studied using bovine fetuin, asialofetuin, IgG, ribonuclease B, and alpha-1 acid glycoprotein (AGP) by PGC-LC-MS. Comprehensive structural isomeric separation of glycopeptides was observed by high-resolution MS and confirmed by MS/MS. The specific structures of the glycopeptide isomers were identified and confirmed through exoglycosidase digestions. Glycosylation analysis of human AGP revealed the potential use of PGC-LC-MS for extensive glycoprotein analysis for biomarker discovery. This newly developed separation technique was shown as a reproducible and useful analytical method to study site-specific isomeric glycosylation.

Original languageEnglish
Pages (from-to)9556-9565
Number of pages10
JournalAnalytical Chemistry
Issue number14
StatePublished - Jul 21 2020


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