Perfluorooctanoic acid for shotgun proteomics.

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 uni...

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Main Authors: Chandra Sekhar Rao Kadiyala, Sara E Tomechko, Masaru Miyagi
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2010-12-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC3012695?pdf=render
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spelling doaj-98d976cd9dee4cfb8de347525ab628d32020-11-25T01:41:55ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-12-01512e1533210.1371/journal.pone.0015332Perfluorooctanoic acid for shotgun proteomics.Chandra Sekhar Rao KadiyalaSara E TomechkoMasaru MiyagiHere, 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.http://europepmc.org/articles/PMC3012695?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Chandra Sekhar Rao Kadiyala
Sara E Tomechko
Masaru Miyagi
spellingShingle Chandra Sekhar Rao Kadiyala
Sara E Tomechko
Masaru Miyagi
Perfluorooctanoic acid for shotgun proteomics.
PLoS ONE
author_facet Chandra Sekhar Rao Kadiyala
Sara E Tomechko
Masaru Miyagi
author_sort Chandra Sekhar Rao Kadiyala
title Perfluorooctanoic acid for shotgun proteomics.
title_short Perfluorooctanoic acid for shotgun proteomics.
title_full Perfluorooctanoic acid for shotgun proteomics.
title_fullStr Perfluorooctanoic acid for shotgun proteomics.
title_full_unstemmed Perfluorooctanoic acid for shotgun proteomics.
title_sort perfluorooctanoic acid for shotgun proteomics.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2010-12-01
description 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.
url http://europepmc.org/articles/PMC3012695?pdf=render
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