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

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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.