Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography

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.