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 18O 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.

Enhanced Energy Metabolism Contributes to the Extended Life Span of Calorie-restricted Caenorhabditis elegans

“…The resulting solution was diluted with 150 l of 100 mM ammonium bicarbonate to reduce PFOA concentration to 0.5%, and the amount of dissolved protein was determined with a DC protein assay kit (Bio-Rad). A total of 1.736 mg of protein in the forward labeling and 1.736 mg of protein in the reverse labeling experiments was digested by Lys-C (1:50 Lys-C to protein ratio w/w) at 37°C for 18 h. The protein digests were dried in a speed-vac concentrator (Savant SC210A, Fisher) at 25°C and then subjected to repeated reconstitution in 100 l of ethanol/ ethyl acetate/water/TFA (0.33:0.33:0.33:0.01, v/v) and evaporation to thoroughly remove PFOA as described previously (31).…”

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

“…These data suggest that endogenous ligands for microglial activation are proteins. Perfluorooctanoic acid (PFOA) was used to solubilize POS proteins for detection of candidate proteins that cause immune reactions by proteomics analysis (45). POS proteins were solubilized by 0.5% PFOA and the insoluble fraction of 0.5% PFOA was further solubilized by 2% PFOA.…”

Enhanced cytotoxicity of pentachlorophenol by perfluorooctane sulfonate or perfluorooctanoic acid in HepG2 cells

“…5B). Like anionic detergents such as sodium dodecyl sulfate, they might preferentially bind plasma membrane fractions (Kudo et al, 2007), and interfere with the membrane/protein interfacial interactions by the membrane solubilization even at concentrations below their critical micelle concentrations (Kragh-Hansen et al, 1998;le Maire et al, 2000;Hu et al, 2003;Kadiyala et al, 2010;Otzen, 2011). For comprehensive understanding the disruptive effects of PFOS (or PFOA) on plasma membrane, further investigations are needed.…”