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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology