Calculated two-dimensional sugar map of pyridylaminated oligosacchardies: Elucidation of the jack bean α-mannosidase digestion pathway of Man₉GlcNAc₂

We have developed a two-dimensional (2-D) mapping of puridylaminated oligosaccharides as an aid to structural determination of glycoprotein-derived oligosaccharides. Using the available data of reverse-phase HPLC of pyridylamino-oligosaccharides, this was further extended to parameterization of unit contribution by each sugar component, which allows the prediction of possible structures from the elution volume. We have extended this approach to the data obtained with amide-silica HPLC column to obtain a calculated 2-D mapping technique for the oligomannose-type oligosacharides (M-series). In this method, the elution volumes of all possible pyridylamino-oligosaccharides up to the size of Glc₁Man₉GlcNAc₂ (50 in total) are calculated from the established UC values to construct a 2-D map. To test the validity of the calculated 2-D map, the structures of intermediate PA-oligosaccharides generated during the α-mannosidase (jack bean) digestion of Man₉GlcNAc₂ (porcine thyroglobulin) were analyzed to establish the digestion pathway. The validity of this approach is substantiated by an independent deduction of the intermediate structures based on structural relationships and the coincidence of elution volumes. Our results agree well with the recently published digestion pathway of Man₅GlcNAc₂ by the same enzyme and that of Man₉GlcNAc₂ by lysosomal α-mannosidase.