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Identification of Glycosylphosphatidylinositol Anchored Proteins and ω-sites Using TiO2-Based Affinity Purification
Glycosylphosphatidylinositol anchored proteins (GPI-APs) in the outer leaflet of the membrane microdomains, commonly referred to as lipid rafts, play important roles in many biological processes. To enable large-scale site-specific analysis of GPI anchoring, we developed a method for identification of ω-sites by mass spectrometry by combining titanium dioxide-based affinity purification and hydrogen fluoride treatment. This method was able to identify ~3-fold more GPI-APs than our previous method: the new technique identified a total of 73 ω-sites derived from 49 GPI-APs. In 13 of the 49 GPI-APs identified, the GPI-anchor attached to multiple amino acids in the C-terminal site, yielding a variety of different protein species. This method allows us to simultaneously identify many GPI-AP protein species with different ω-sites.
Sample Processing Protocol
The membrane fraction of cell lysates was resuspended in 50 mM HEPES [pH 7.5] and 1% (v/v) Triton X-114. This solution was subjected to Triton X-114 two-phase separation and PI-PLC treatment. Supernatants were concentrated by TCA/acetone precipitation and subjected to in-solution digestion (trypsin or chymotrypsin). The GPI-anchored peptide enrichment procedure was carried out using Titansphere Phos-TiO Kit. The bound GPI-anchored peptides were eluted with 5% NH3–H2O. The eluates were immediately dried under vacuum, and treated with aqueous hydrogen fluoride for 12 h at 4°C to cleave the GPI-anchor ethanolamine–phosphate bond. The samples were completely dried under vacuum and dissolved in 0.1% TFA for mass-spectrometric analysis.
Data Processing Protocol
All MS/MS data were analyzed using Proteome Discoverer (v.126.96.36.1999, Thermo Fisher Scientific), applying Mascot (v.2.4.0, Matrix Science) for peptide identification. The data were queried against a UniProt/SWISS-PROT database (v2012-0711; Homo sapiens 20,232 sequences). All database searches were performed using a precursor mass tolerance of ±5 ppm, fragment ion mass tolerance of ±0.5 Da, enzyme name set to semi specific (trypsin or chymotrypsin), and a maximum missed-cleavages value of 2. For the in-solution digestion procedure, variable modifications were specified as oxidation of Met. Fixed modifications were specified as carboxymethyl of Cys. To identify GPI-anchored peptide sequences, the EtN moiety (+43.0422 Da) was set as a variable modification of the C-terminal peptide.
Yusuke Masuishi, Yokohama City University
Yusuke Masuishi, Yusuke Masuishi, Yokohama City University Hisashi Hirano, Graduate School of Medical Life Science and Advanced Medical Research Center Yokohama City University, Yokohama Kanagawa, Japan ( lab head ) ( lab head )
Masuishi Y, Kimura Y, Arakawa N, Hirano H. Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines. Data Brief. 2016 Apr 8;7:1302-5. eCollection 2016 Jun PubMed: 27141528
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