title: Perfluorooctanoic acid for shotgun proteomics.
abstract: Here, we describe the novel use of a volatile surfactant, perfluorooctanoic acid (PFOA), for shotgun proteomics. PFOA was found to solubilize membrane proteins as effectively as sodium dodecyl sulfate (SDS). PFOA concentrations up to 0.5% (w/v) did not significantly inhibit trypsin activity. The unique features of PFOA allowed us to develop a single-tube shotgun proteomics method that used all volatile chemicals that could easily be removed by evaporation prior to mass spectrometry analysis. The experimental procedures involved: 1) extraction of proteins in 2% PFOA; 2) reduction of cystine residues with triethyl phosphine and their S-alkylation with iodoethanol; 3) trypsin digestion of proteins in 0.5% PFOA; 4) removal of PFOA by evaporation; and 5) LC-MS/MS analysis of the resulting peptides. The general applicability of the method was demonstrated with the membrane preparation of photoreceptor outer segments. We identified 75 proteins from 1 µg of the tryptic peptides in a single, 1-hour, LC-MS/MS run. About 67% of the proteins identified were classified as membrane proteins. We also demonstrate that a proteolytic (18)O labeling procedure can be incorporated after the PFOA removal step for quantitative proteomic experiments. The present method does not require sample clean-up devices such as solid-phase extractions and membrane filters, so no proteins/peptides are lost in any experimental steps. Thus, this single-tube shotgun proteomics method overcomes the major drawbacks of surfactant use in proteomic experiments.
authors.affiliation: Case Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Chandra Sekhar Rao
authors.initials: CS
authors.lastname: Kadiyala
authors.forename: Sara E
authors.initials: SE
authors.lastname: Tomechko
authors.forename: Masaru
authors.initials: M
authors.lastname: Miyagi
mesh_terms: D000818:Animals; D002210:Caprylates / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D003553:Cystine / Q000737:chemistry; D000431:Ethanol / Q000737:chemistry; D005466:Fluorocarbons / Q000378:metabolism*; D008954:Models, Biological; D010103:Oxygen Isotopes / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010720:Phosphines / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D013501:Surface-Active Agents / Q000378:metabolism; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000737:chemistry
chemical_list: D002210:Caprylates; D005466:Fluorocarbons; D010103:Oxygen Isotopes; D010455:Peptides; D010720:Phosphines; D013501:Surface-Active Agents; D000431:Ethanol; D003553:Cystine; C023036:perfluorooctanoic acid; D014357:Trypsin