Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS.
Both organic solvent and surfactant have been used for dissolving membrane proteins for shotgun proteomics. In this work, two methods of protein solubilization, namely using 60% methanol or 1% SDS, to dissolve and analyze the inner membrane fraction of an Escherichia coli K12 cell lysate were compared. A total of 358 proteins (1417 unique peptides) from the methanol-solubilized protein mixture and 299 proteins (892 peptides) from the SDS-solubilized sample-were identified by using trypsin digestion and 2-D LC-ESI MS/MS. It was found that the methanol method detected more hydrophobic peptides, resulting in a greater number of proteins identified, than the SDS method. We found that 159 out of 358 proteins (44%) and 120 out of 299 proteins (40%) detected from the methanol- and SDS-solubilized samples, respectively, are integral membrane proteins. Among the 190 integral membrane proteins 70 were identified exclusively in the methanol-solubilized sample, 89 were identified by both methods, and only 31 proteins were exclusively identified by the SDS method. It is shown that the integral membrane proteins reflected the theoretical proteome for number of transmembrane helices, length, functional class, and topology, indicating there was no bias in the proteins identified.
Zhang
Nan
N
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Chen
Rui
R
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Young
Nelson
N
Department of Biological Sciences and Computing Science, University of Alberta, Edmonton, Alberta, Canada.
Wishart
David
D
Department of Biological Sciences and Computing Science, University of Alberta, Edmonton, Alberta, Canada.
Winter
Philip
P
Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta, Canada.
Weiner
Joel H
JH
Institute for Biomolecular Design, University of Alberta, Edmonton, Alberta, Canada.|Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Li
Liang
L
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
D002853:Chromatography, Liquid; D048168:Escherichia coli K12 / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D000432:Methanol / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D020543:Proteome; D012967:Sodium Dodecyl Sulfate; D000432:Methanol