Structural and Functional analyses of GPI-anchored proteins by nano-LC/MS

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Glycosylphosphatidylinositol (GPI)-anchored proteins existing in brain membrane play an essential role in constitution of nerve network system. Some reports suggest that glycosylation of GPI-anchored proteins could alter during the embryo development, however, the carbohydrate function and structure of only a few GPI-anchored proteins are reported due to the difficulty of their purification and analysis. In this project we studied site-specific glycosylation of GPI-anchored proteins in rat brain by the separation with SDS-PAGE followed LC/Ms^n.GPI-linked proteins, which were released by PIPLC treatment from the rat brain membrane, were fractionated and separated by SDS-PAGE. The bands at 20-25 kDa and 45-85 kDa were identified as Thy-1 and a mixture of LAMP, OBCAM, NTM, kilon, respectively. First, Thy-1 was extracted from the gel with 1% SDS, and the tryptic digest of Thy-1 was subjected to LC/MS^n for site-specific glycosylation analysis. It was confirmed that Asn23 and 98 are occupied with high-mannose type, hybrid and complex type oligosaccharides, and Asn74 was attached to fucosylcomplex type oligosaccharides. Likewise, site-specific glycosylation analysis of LAMP, OBCAM, NTM, and kilon were carried out by extraction of the proteins from the band at 45-85 kDa followed by LC/MS^n. We demonstrated that the glycosylation sites in the first-domain are occupied with high-mannose type oligosaccharide among four proteins, and those in the third-domain are attached to oligosaccharides bearing Le^x motif.We are planning to apply this method to the site-specific glycosylation analysis of other GPI-binding proteins.