sodium-dodecyl-sulfate and benzoylarginine-ethyl-ester

The hydrophobic nature of most membrane proteins severely complicates their extraction, proteolysis and identification. Although detergents can be used to enhance the solubility of the membrane proteins, it is often difficult for a detergent not only to have a strong ability to extract membrane proteins, but also to be compatible with the subsequent proteolysis and mass spectrometric analysis. In this study, we made evaluation on a novel application of sodium laurate (SL) to the shotgun analysis of membrane proteomes. SL was found not only to lyse the membranes and solubilize membrane proteins as efficiently as SDS, but also to be well compatible with trypsin and chymotrypsin. Furthermore, SL could be efficiently removed by phase transfer method from samples after acidification, thus ensuring not to interfere with the subsequent CapLC-MS/MS analysis of the proteolytic peptides of proteins. When SL was applied to assist the digestion and identification of a standard protein mixture containing bacteriorhodoposin and the proteins in rat liver plasma membrane-enriched fractions, it was found that, compared with other two representative enzyme- and MS-compatible detergents RapiGest SF (RGS) and sodium deoxycholate (SDC), SL exhibited obvious superiority in the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.

Topics: Animals; Arginine; Cell Membrane; Chymotrypsin; Computational Biology; Deoxycholic Acid; Detergents; Lauric Acids; Liver; Mass Spectrometry; Peptide Hydrolases; Proteomics; Rats; Sodium Dodecyl Sulfate; Tandem Mass Spectrometry; Trypsin

Improved in-solution tryptic digestion of proteins in terms of speed and peptide coverage was achieved with the aid of a novel acid-labile anionic surfactant (ALS). Unlike SDS, ALS solubilizes proteins without inhibiting trypsin or other common endopeptidases activity. Trypsin activity was evaluated in the presence of various denaturants; little or no decrease in proteolytic activity was observed in 0.1-1% ALS solutions (w/v). Sample preparation prior to mass spectrometry and liquid chromatography analysis consists of sample acidification. ALS degrades rapidly at low-pH conditions, which eliminates surfactant-caused interference with analysis. Described methodology combines the advantages of protein solubilization, rapid digestion, high peptide coverages, and easy sample preparation for mass spectrometry and liquid chromatography analyses.

Topics: Alkanesulfonates; Angiotensin II; Arginine; Bacteriorhodopsins; Chromatography, High Pressure Liquid; Chymotrypsin; Dioxolanes; Endopeptidases; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Molecular Structure; Muramidase; Myoglobin; Neurotensin; Ovalbumin; Proteins; Sodium Dodecyl Sulfate; Solubility; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface-Active Agents; Trypsin; Ubiquitin; Urea