Development of the method for high-sensitivity structural analysis of carbohydrate moieties of a glycoprotein by mass spectrometry

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In order to establish the method for high-sensitivity structural analysis of carbohydrate moieties of a glycoprotein, the following two approaches have been examined: 1 ) electrospray-ionization (ESI) tandem mass spectrometry (MS/MS) has been directly applied for the analysis of glycopeptides with relatively large molecular masses (3000 Da 〜), which were obtained by an in-gel digestion of a glycoprotein. 2) the chemical derivatization with 4-aminobenzoic acid 2-diethylaminoethyl ester (ABDEAE), which was developed for the high-sensitivity detection and structural analysis of carbohydrates by MS, has been applied to carbohydrates cleaved from a glycoprotein with PNGase F. As the results obtained for human transferrin 1) the two glycopeptides, which were produced by in-gel digestion with lysylendopeptidase and separated by HPLC, gave specific fragmentation at glycosidic bonds upon low-energy collision induced dissociation in ESI-MS/MS. It could clearly reveal the sequence and partial branching structure of the carbohydrate moieties. 2) the carbohydrate moieties derived from transferrin were derivatized in gel with ABDEAE. The excess reagent was found to be efficiently removed by passing a reaction mixture through a polymer-based amino-propyl resin In addition, the salts were separated from the carbohydrate derivatives by a reversed-phase resin. Consequently, the derivatives could be subjected to on-line micro-HPLC/ESI-MS, which allowed the detection of such those prepared from the gel band corresponding to ca. 10 pmol of transferrin as a charging amount to SDS-PAGE."SeqMS", a software aid for de novo sequencing by MS/MS, has been modified so as to efficiently interpret MS/MS spectra of both glycopeptides and carbohydrate derivatives.