New method for determining the sugar composition of glycoproteins, glycolipids, and oligosaccharides by high-performance liquid chromatography

A new method is reported that can be performed within a single vessel to an alyze the composition of aldose, hexosamine, and sialic acid residues of gl ycoproteins, glycolipids, and oligosaccharides, Glycoconjugates are treated with sialidase or subjected to mild acid hydrolysis, before being treated with N-acetylneuraminic acid aldolase to convert the free sialic acid resid ues to their corresponding N-acylmannosamines, The reaction mixture is then successively subjected to acid hydrolysis tin order to produce monosacchar ides), N-acetylation, and conversion with paminobenzoic acid ethyl ester (A BEE), The ABEE-converted monosaccharides are simultaneously determined by r everse-phase high-performance liquid chromatography, Determination of the s ugar compositions of bovine fetuin, II(3)NeuGc alpha-LacCer, and 3'-sialyll actose with this method was found to be highly accurate. Linearity of the p eak area vs. the amount of bovine fetuin ranged from 1. to 50 mu g in all A BEE-converted monosaccharides. With a slight modification to this method, s ialic acid residues can be separately determined as NeuAc and NeuGc, This n ovel method and its modified version are used to demonstrate the sugar comp ositions of alpha(1)-acid glycoproteins from several sources.