The role of acid labile surfactant-I in protein extraction from sodium dodecyl sulfate polyacrylamide gels
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The surfactant known as acid labile surfactant I (ALS-I) (RapiGest) was investigated to probe its effectiveness in enhancing protein digestion and subsequent protein identification and to determine its suitability for use in whole protein extraction methods. Tryptic solution and in-gel digests of several standard proteins showed a distinct increase in the number of peptides matched in ALS-I aided digests versus digests performed without ALS-I. Several experiments gave no identification unless ALS-I was present. The ability to analyse whole protein samples with detergent or extracted from gels has eluded scientists and slowed certain areas of research such as the investigation and analysis of membrane proteins. ALS-I was ascertained to be compatible with and enhance whole protein extractions from sodium dodecyl sulfate-polyacrylamide gels (SDS-PA). Extraction was optimized by testing a series of extraction solutions containing ALS-I, DTT and tris/glycine. Extraction efficiency was shown to vary with incubation time and larger proteins proved harder to extract from a gel than smaller proteins. Subsequent analysis of protein extracts was performed by matrix-assisted laser desorption/ionisation mass spectrometry and liquid chromatography-electrospray ionisation mass spectrometry. In order to analyse ALS-I extracted protein by MALDI-TOF MS it was necessary to develop a novel slow crystallisation using neutral matrix systems and sample preparation techniques. The neutral matrices 2-amino-4-methyl-5-nitropyridine, 4- nitroaniline and 6-aza-2-thiothymine gave good whole protein signals at M+H and M+2H. Whole protein extracts from SDS-polyacrylamide gels were also analysed by LC-ESI MS, giving good spectra for a range of proteins from superoxide dismutase (SOD) to bovine serum albumin (BSA). Despite small mass inaccuracies, the novel possibility of analysing whole protein extracts from SDS-polyacrylamide gels containing detergent and sufficient protein to give clear protein spectra by LC-ESI MS is very promising, and progress in this technique may prove invaluable to further research into the proteome.
Thesis, MPhil Master of Philosophy
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