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Application of two-dimensional gel electrophoresis and LC/MS/MS for the structural and functional analysis of carbohydrates in glycoproteins

二维凝胶电泳和 LC/MS/MS 在糖蛋白中碳水化合物结构和功能分析中的应用

基本信息

批准号：
12672102
负责人：
KAWASAKI Nana
金额：
$ 2.05万
依托单位：
National Institute of Health Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

For the functional and structural analyses of glycoproteins expressed in tissues and cells, we have developed the glycoprotein profiling by two-dimensional gel electrophoresis (2D-GE)followed by oligosaccharide profiling and glycopeptide mapping by LC/MS.First, using hepatocyte growth factor, thrombomodulin, and tissue plasminogen activator as model glycoproteins, we developed the oligosaccharide profiling which can be used for the analyses of distribution and structure of oligosaccharides released from glycoproteins. Protein moieties were used for identification by LC/MS/MS after tryptic digestion. We also developed the glycopeptide mapping which can elucidate the glycosylation sites and site-specific glycosylation by drawing out the glycopeptides from mixture of peptides and glycopetides.Second, we applied the 2D-GE, oligosaccharide profiling and glycopeptide mapping to the glycoprotein profiling followed by structural analysis of in-gel glycoproteins. GPI anchor proteins were prepared by Triton X114 and PIPLC treatment from rat brains, and their profile was drawn by 2D-GE. In-gel proteins were digested with PNGase F and trypsin. Using tryptic digest, these proteins were identified to NCAM, LAMP, NTM, OBCAM, kilon, and prion. Oligosaccharides in these proteins were characterized to BA-2, high-mannose type, and sialylated fucosyl complex type oligosaccharides. The presented method can be used for functional and structural analysis of glycoproteins in cells and tissues.

对于组织和细胞中表达的糖蛋白的功能和结构分析，我们已经开发了通过二维凝胶电泳（2D-GE）的糖蛋白谱，随后通过LC/MS的寡糖谱和糖肽谱。首先，使用肝细胞生长因子、血栓调节蛋白和组织纤溶酶原激活剂作为模型糖蛋白，我们开发了寡糖谱分析法，可用于分析糖蛋白释放的寡糖的分布和结构。蛋白质部分用于胰蛋白酶消化后通过LC/MS/MS进行鉴定。其次，我们将2D-GE、寡糖谱和糖肽图谱技术应用于糖蛋白谱分析，并对凝胶内糖蛋白进行了结构分析。用TritonX 114和PIPLC处理大鼠脑组织制备GPI锚蛋白，用2D-GE绘制其图谱。用PNGase F和胰蛋白酶消化凝胶内蛋白。使用胰蛋白酶消化，这些蛋白质被鉴定为NCAM、LAMP、NTM、OBCAM、kilon和朊病毒。这些蛋白中的寡糖被表征为BA-2、高甘露糖型和唾液酸化岩藻糖基复合物型寡糖。所提出的方法可用于细胞和组织中糖蛋白的功能和结构分析。