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

为了对组织和细胞中表达的糖蛋白进行功能和结构分析，我们开发了二维凝胶电泳（2D-GE）的糖蛋白谱分析，然后是低聚糖谱分析和LC/MS的糖肽图谱分析。首先，我们以肝细胞生长因子、血栓调节蛋白和组织纤溶酶原激活物为模型糖蛋白，建立了寡糖谱，用于分析糖蛋白释放的寡糖分布和结构。蛋白质部分经胰酶切后采用LC/MS/MS鉴定。我们还开发了糖肽定位，通过从多肽和糖肽的混合物中提取糖肽来阐明糖基化位点和位点特异性糖基化。其次，我们将2D-GE、低聚糖图谱和糖肽图谱应用于糖蛋白图谱，然后对凝胶内糖蛋白进行结构分析。用Triton X114和PIPLC处理大鼠脑制备GPI锚定蛋白，并用2D-GE绘制其结构谱。凝胶蛋白用PNGase F和胰蛋白酶消化。用胰消化法鉴定这些蛋白为NCAM、LAMP、NTM、OBCAM、kilon和prion。这些蛋白中的低聚糖被鉴定为BA-2型、高甘露糖型和唾液化聚焦基复合物型低聚糖。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.