Development of the method for the analysis of carbohydrates derived from glycoproteins at the level detected by silver staining method

碳水化合物以及核酸和蛋白质是糖蛋白、糖脂和蛋白聚糖等糖缀合物的主要成分。糖缀合物碳水化合物链合成的相关酶起着重要作用，其生物合成受基因组的严格调控。然而，碳水化合物是由糖基转移酶和水解酶等酶协同合成的，具有内在的异质性。应该注意的是，糖缀合物的异质性和碳水化合物链的数量随着生物事件而改变。此外，碳水化合物在细胞-细胞识别中起着相当重要的作用。虽然基因组学和蛋白质组学研究取得了一定的进展，但糖缀合物的分析还需要新的方法。目前，我们采用二维平板凝胶电泳进行蛋白质组分析。凝胶上分离的蛋白质带通常通过银染色和/或荧光标记方法可见，以便在fmol水平下进行检测。检测到的蛋白质可以通过蛋白质测序仪或质谱分析。但是，由于碳水化合物链的结构复杂，并且即使在单个蛋白质分子中也存在各种碳水化合物链，因此目前的技术还无法分析蛋白质核心上的碳水化合物链。虽然我们可以在pmol水平上分析碳水化合物链，但要分析二维凝胶上糖蛋白点上的碳水化合物链，我们需要实现更高灵敏度的检测。在本项目中，我们的目标是在化学或酶法释放的碳水化合物链进行荧光标记后，利用毛细管电泳激光诱导荧光检测在原子醇水平上确定糖蛋白的碳水化合物链。这意味着我们必须分析银染色法检测到的糖蛋白斑点的碳水化合物链。

Carbohydrates as well as nucleic acids and proteins are major conctituents of glycoconjugates such as glycoproteins, glycolipids and proteoglycans. Related enzymes for the synthesis of carbohydrate chains of glycoconjugates play important roles, and their biosyntheses are strictly regulated by genome. However, carbohydrates are synthesized by collaborative works of the enzymes such as glycosyl transferases and hydrolases, and have intrinsic heterogeneity. It should be noted that heterogeneity and the amount of carbohydrate chains in glyconjugates are changed with biological events. Furthermore, carbohydrates play quite important roles for cell-cell recognitions. Progress in genome and proteomics project has been achieved, but we have to novel strategies for the analysis of glycoconjugates.At present, we employ two dimensional slab gel electrophoresis for the proteome analysis. The separated protein band on the gel are often visualized by silver staining and/or fluorescent-labeling methods to allow detection at fmol level. The detected protein can be analyzed by a protein sequencer or mass spectrometry. But carbohydrate chains attached to the protein core can not be analyzed by the present technology due to the complex structures and the presence of various carbohydrate chains even in a single molecule of a protein. Although we can analyze carbohydrate chains at pmol level, we have to achieve higher sensitive detection to analyze the carbohydrate chains in a glycoprotein spot on 2D gel.In this project, we focused the objective to determine the carbohydrate chains of a glycoprotein at attomol level using capillary electrophoresis with laser-induced fluorescent detection after fluorescent labeling of carbohydrate chains released by chemical or enzymatic method. This means that we have to analyze the carbohydrate chains of the glycoprotein spot detected by silver staining method.