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

• Published in: Archives of biochemistry and biophysics 1999-03, Vol.363 (1), p.60-67
• Main Authors: Taguchi, Ryo, Yamazaki, Junko, Takahashi, Mayumi, Hirano, Akiko, Ikezawa, Hiroh
• Format: Article
• Language: English
• Subjects: Acetylcholinesterase - metabolism; Animals; Carbohydrate Sequence; Cattle; Chemical Fractionation; Chromatography, Ion Exchange; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane - chemistry; Erythrocyte Membrane - enzymology; ESI–MS; Gas Chromatography-Mass Spectrometry; GC–MS; Glycosides - chemistry; Glycosylphosphatidylinositols - chemistry; GPI anchor; Inositol - chemistry; Inositol - metabolism; inositol phosphoglycan; Isomerism; MALDI-TOF–MS; Molecular Sequence Data; Peptides - chemistry; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; posttranslational processing; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Type C Phospholipases - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1998.1054

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.