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

银染法检测糖蛋白来源碳水化合物的分析方法的开发

基本信息

批准号：
14370732
负责人：
KAKEHI Kazuaki
金额：
$ 4.16万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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.

糖蛋白、糖脂、蛋白聚糖等糖复合物的主要成分是碳水化合物、核酸和蛋白质。糖复合物中糖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作用合成的，具有内在的异质性。应当注意的是，糖缀合物中的异质性和糖链的量随着生物事件而改变。此外，碳水化合物在细胞间的相互作用中起着非常重要的作用。基因组和蛋白质组学的研究取得了很大的进展，但糖缀合物的分析方法还有待改进，目前蛋白质组分析主要采用双向平板凝胶电泳技术。凝胶上分离的蛋白质条带通常通过银染色和/或荧光标记方法可视化，以允许在fmol水平下检测。可通过蛋白质测序仪或质谱法分析检测到的蛋白质。但是，由于复杂的结构和甚至在蛋白质的单个分子中存在各种碳水化合物链，连接到蛋白质核心的碳水化合物链不能通过现有技术进行分析。虽然我们可以在pmol水平上分析糖链，但我们必须实现更高灵敏度的检测来分析2D凝胶上糖蛋白斑点中的糖链。在本项目中，我们的目标是在荧光标记化学或酶法释放的糖链后，使用毛细管电泳-激光诱导荧光检测来在阿托摩尔水平上确定糖蛋白的糖链。这意味着我们必须分析通过银染法检测的糖蛋白斑点的糖链。