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 analyze the composition of aldose, hexosamine, and sialic acid residues of glycoproteins, 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 residues to their corresponding N-acylmannosamines. The reaction mixture is then successively subjected to acid hydrolysis (in order to produce monosaccharides), N-acetylation, and conversion with p-aminobenzoic acid ethyl ester (ABEE). The ABEE-converted monosaccharides are simultaneously determined by reverse-phase high-performance liquid chromatography. Determination of the sugar compositions of bovine fetuin, II3NeuGc alpha-LacCer, and 3'-sialyllactose with this method was found to be highly accurate. Linearity of the peak area vs. the amount of bovine fetuin ranged from 1 to 50 micrograms in all ABEE-converted monosaccharides. With a slight modification to this method, sialic acid residues can be separately determined as NeuAc and NeuGc. This novel method and its modified version are used to demonstrate the sugar compositions of alpha 1-acid glycoproteins from several sources.