title: Mass spectrometric identification of glycosylphosphatidylinositol-anchored peptides.
abstract: Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs.
authors.affiliation: Graduate School of Medical Life Science and ‡Advanced Medical Research Center, Yokohama City University , Yokohama, Kanagawa 236-0004, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Masuishi
authors.forename: Ayako
authors.initials: A
authors.lastname: Nomura
authors.forename: Akiko
authors.initials: A
authors.lastname: Okayama
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kimura
authors.forename: Noriaki
authors.initials: N
authors.lastname: Arakawa
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hirano
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D058851:GPI-Linked Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D010446:Peptide Fragments; C508196:SMPDL3B protein, human; D013108:Sphingomyelin Phosphodiesterase