Identification of a New Glycosylphosphatidylinositol-Anchored 42-kDa Protein and Its C-Terminal Peptides from Bovine Erythrocytes by Gas Chromatography–, Time-of-Flight–, and Electrospray-Ionization–Mass Spectrometry

By treatment with phosphatidylinositol-specific phospholipase C (PIPLC), we obtained several candidates of glycosylphosphatidylinositol (GPI)-anchored proteins such as 55, 42, 40, and 30 kDa from bovine erythrocyte membrane, in addition to the well-known GPI-anchored protein acetylcholinesterase. In these proteins, the presence ofmyo-inositol was confirmed by gas chromatography (GC)–mass spectrometry. Among them, the 42-kDa protein was further analyzed by electrospray-ionization (ESI)–mass spectrometry (MS) after hydrolysis by lysyl endoprotease. By liquid chromatography (LC)–ESI–MS analysis, C-terminal peptides bearing the products of GPI (Ct. GPI-peptides) were effectively detected by combination with in-source collision and multifunctional scanning for the several characteristic fragment ions from the GPI-anchor structure. Existence of microheterogeneity was also observed in the Ct. GPI-peptides from the 42-kDa protein. This result was confirmed by analysis with time-of-flight (TOF)–MS. Furthermore, one of the Ct. GPI-peptides was analyzed in ESI–MS–MS mode. Characteristic fragment ions were effectively detected by collision-induced decay. By the result of MS–MS analysis, this GPI-anchor structure was revealed to contain additionalN-acetyl hexosamine. By the above-mentioned method, the C-terminal GPI-anchor structure can be easily identified from the target protein even if its amino acid sequence data are not available.