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

为了建立糖蛋白碳水化合物结构的高灵敏度分析方法，研究了以下两种方法：1)将电喷雾电离（ESI）串联质谱（MS/MS）直接应用于糖蛋白凝胶内酶切得到的分子量较大（3000 Da ~）的糖肽的分析。2)利用质谱法对碳水化合物进行高灵敏度检测和结构分析而建立的4-氨基苯甲酸2-二乙基氨基乙酯（ABDEAE）化学衍生化反应，已应用于PNGase f对糖蛋白裂解的碳水化合物的分析。在ESI-MS/MS中，在低能碰撞诱导解离时，糖苷键出现了特定的断裂。它能清晰地揭示碳水化合物的序列和部分分支结构。2)用ABDEAE凝胶化转铁蛋白衍生的碳水化合物部分。通过聚合物基氨基丙基树脂的反应混合物可以有效地去除多余的试剂。此外，通过反相树脂将盐与碳水化合物衍生物分离。因此，这些衍生物可以通过在线微高效液相色谱/ESI-MS进行检测，从而可以从相当于约10 pmol转铁蛋白作为SDS-PAGE充电量的凝胶带中检测到这些衍生物。“SeqMS”是一款MS/MS从头测序软件，它可以有效地解释糖肽和碳水化合物衍生物的MS/MS光谱。

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.