Analysis of Oligosaccharide Structures from the Reducing End Terminal by Combining Partial Acid Hydrolysis and a Two-Dimensional Sugar Map

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Structure analysis is a necessary step in elucidating the roles of glycoprotein sugar chains. Glycoproteins with biological activity isolated from natural sources, however, are often obtainable in only limited amounts. To circumvent this drawback, we developed a fluorescence labeling method, which we have been using in combination with exoglycosidase digestion. Although the method is useful, we frequently encounter difficulty in obtaining enzymes or in the incubation conditions, and even when we do have an enzyme, it is not always able to liberate sugar residues due to substrate specificity.To overcome this, a new sensitive and convenient method for the structural analysis of oligosaccharides was developed, in which partial acid hydrolysis was combined with two-dimensional sugar mapping of pyridylamino (PA-) oligosaccharides. APA-oligosaccharide was partially hydrolyzed, the acid hydrolysis conditions being optimized so as to obtain various PA-olgosaccharide fragments with high yields from different types of PA-oligosaccharides. The acid hydrolyzates were then fractionated every 1 glucose unit by size-fractionation HPLC, and each fraction was analyzed by reversed-phase HPLC. The structure of each PA-oligosaccharide fragment was identified on a two-dimensional map prepared with standard PA-sugar chains, after which the original PA-oligosaccharide was reconstructed from the reducing end terminal based on the obtained structures of the PA-oligosaccharide fragments. The method was successfully applied to resolving the structure of an N-linked sugar chain.