Kotani N, Takasaki S
Department of Biochemistry, University of Tokyo, Japan.
Anal Biochem. 1997 Oct 1;252(1):40-7. doi: 10.1006/abio.1997.2300.
To apply high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to the accurate and sensitive analysis of O-linked oligosaccharide alditol, removal of glycopeptides and peptides from oligosaccharide samples obtained by beta-elimination was first investigated. The results obtained by using fetuin as a model glycoprotein indicated that (glyco)peptides derivatized by fluorescamine are easily removed from the beta-eliminated oligosaccharide sample by means of a Sep-Pak C18 cartridge due to their increased hydrophobicity. This clean-up procedure was also effectively applied to the beta-eliminated oligosaccharide sample obtained from delipidated sheep erythrocyte ghosts. Furthermore, structural analysis of the oligosaccharide alditols by HPAEC-PAD was successfully performed in combination with partial and complete desialylation, composition analysis, periodate oxidation, and glycosidase digestion and revealed that sheep erythrocyte membrane glycoproteins contain Gal beta 1-->3Gal-NAc and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->3GalNac, to which one or two residues of Neu5Ac and Neu5Gc are attached in different combinations.
为了将高pH值阴离子交换色谱-脉冲安培检测法（HPAEC-PAD）应用于O-连接寡糖糖醇的准确和灵敏分析，首先研究了从β-消除法获得的寡糖样品中去除糖肽和肽的方法。以胎球蛋白作为模型糖蛋白所获得的结果表明，由于其疏水性增加，经荧光胺衍生化的（糖）肽可通过Sep-Pak C18柱轻易地从β-消除的寡糖样品中去除。该净化程序也有效地应用于从脱脂羊红细胞血影获得的β-消除的寡糖样品。此外，结合部分和完全脱唾液酸、组成分析、高碘酸盐氧化和糖苷酶消化，成功地利用HPAEC-PAD对寡糖糖醇进行了结构分析，结果显示羊红细胞膜糖蛋白含有Galβ1→3Gal-NAc和Galβ1→4GlcNAcβ1→3Galβ1→3GalNac，其上以不同组合连接有一个或两个Neu5Ac和Neu5Gc残基。
