Takegawa Yasuhiro, Deguchi Kisaburo, Ito Hiroki, Keira Takuro, Nakagawa Hiroaki, Nishimura Shin-Ichiro
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.
J Sep Sci. 2006 Nov;29(16):2533-40. doi: 10.1002/jssc.200600133.
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.
天冬酰胺连接的寡糖（N-聚糖）通常表现出结构异质性，尤其是在具有唾液酸化N-聚糖的蛋白质中，因此，其结构分析仍然非常困难。一种带有磺基甜菜碱官能团的两性离子型亲水相互作用色谱柱（称为ZIC-HILIC柱）被应用于α-1-酸性糖蛋白胰蛋白酶解肽段的分离。结果表明，ZIC-HILIC分离柱对唾液酸化N-糖肽具有选择性，并且基于对唾液酸化N-聚糖异构体以及先前报道的中性N-聚糖和N-糖肽的结构识别具有高分离能力。用2-氨基吡啶衍生化的中性和唾液酸化N-聚糖（PA N-聚糖）的保留特性表明，N-聚糖的保留主要基于与ZIC-HILIC柱表面产生的富水液层的亲水相互作用。此外，被抗衡离子屏蔽的静电排斥相互作用有效地调节了唾液酸化N-聚糖异构体的分离和识别。
