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 of myo-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 additional N-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.
Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya City, Aichi, 467, Japan. taguchi@phar.nagoya-cu.ac.jp
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Taguchi
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Yamazaki
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Takahashi
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Hirano
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Ikezawa
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000201:enzymology; D008401:Gas Chromatography-Mass Spectrometry; D006027:Glycosides / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D007294:Inositol / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D010738:Type C Phospholipases / Q000378:metabolism
D006027:Glycosides; D017261:Glycosylphosphatidylinositols; D010455:Peptides; D007294:Inositol; D000110:Acetylcholinesterase; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase