title: Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography.
abstract: Asparagine-linked oligosaccharides (N-glycans) usually show structural heterogeneity, especially in proteins with sialylated N-glycans and, therefore, their structural analysis is still very difficult. A zwitterionic type of hydrophilic interaction chromatography column with sulfobetaine functional groups (called a ZIC-HILIC column) was applied to the separation of tryptic peptides of alpha-1-acid glycoprotein. It was demonstrated that the ZIC-HILIC separation column has a selectivity for sialylated N-glycopeptides and a high capability for separation based on the structural recognition of sialylated N-glycan isomers as well as for the previously reported neutral N-glycans and N-glycopeptides. The retention characteristics of neutral and sialylated N-glycans derivatized with 2-aminopyridine (PA N-glycans) demonstrate that the retentions of the N-glycans are based primarily on hydrophilic interaction with the water-rich liquid layer generated on the surface of the ZIC-HILIC column. In addition, the electrostatic repulsion interaction shielded with counter ions effectively tunes the separation and recognition of sialylated N-glycan isomers.
authors.affiliation: Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takegawa
authors.forename: Kisaburo
authors.initials: K
authors.lastname: Deguchi
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ito
authors.forename: Takuro
authors.initials: T
authors.lastname: Keira
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000631:Aminopyridines / Q000032:analysis / Q000737:chemistry; D001622:Betaine / Q000031:analogs & derivatives / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity; D013997:Time Factors
chemical_list: D000631:Aminopyridines; D006020:Glycopeptides; D009844:Oligosaccharides; D010455:Peptides; D011134:Polysaccharides; D001622:Betaine; C483727:sulfobetaine; D019158:N-Acetylneuraminic Acid; C032439:alpha-aminopyridine