Analysis of O-linked oligosaccharide alditols by high-pH anion-exchange chromatography with pulsed amperometric detection.

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.