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

为了分析在组织和细胞中表达的糖蛋白的功能和结构，我们建立了二维凝胶电泳法(2D-GE)糖蛋白图谱和LC/MS糖肽图谱。首先，我们以肝细胞生长因子、血栓调节蛋白和组织型纤溶酶原激活物为模型糖蛋白，建立了可用于分析糖蛋白释放的寡糖的分布和结构的寡糖图谱。胰酶消化后，用LC/MS/MS鉴定蛋白质组分。其次，我们将2D-GE、寡糖图谱和糖肽图谱应用到糖蛋白图谱中，并对凝胶内糖蛋白进行了结构分析。用Triton X114和PIPLC处理大鼠脑内制备GPI锚定蛋白，并用2D-GE绘制其图谱。用PNGase F和胰酶消化凝胶内蛋白。经胰酶消化鉴定，这些蛋白分别为NCAM、LAMP、NTM、OBCAM、KYON和Prion。这些蛋白质中的寡糖被鉴定为BA-2、高甘露糖型和唾液酸岩藻糖复合型低聚糖。该方法可用于细胞和组织中糖蛋白的功能和结构分析。

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.