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

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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.