title: Comparison of surfactant-assisted shotgun methods using acid-labile surfactants and sodium dodecyl sulfate for membrane proteome analysis.
abstract: Three surfactant-assisted shotgun methods using acid labile surfactants, sodium-3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)-methoxyl]-1-propanesulfonate (RapiGest) and 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), and sodium dodecyl sulfate (SDS) were investigated for their applicability to membrane proteome analysis. It is shown that RapiGest is a preferred reagent for handling membrane proteomes of Escherichia coli and MCF7 cells for liquid chromatography tandem mass spectrometry (LC MS/MS) analysis of tryptic digests. The RapiGest method allowed identification of more peptides and proteins than the SDS and PPS methods and there was no apparent bias for the type of peptides and proteins identified by the RapiGest and SDS methods, while a slightly higher proportion of hydrophilic peptides and proteins were identified by the PPS method. The performance of the SDS and PPS methods is similar in terms of the numbers of peptides and proteins identified. Since the SDS method required the removal of SDS using a technique such as strong-cation exchange (SCX), we further investigated the effect of SCX on sample loss through analyzing the digest of an enriched E. coli membrane fraction as well as a standard protein, bovine serum albumin (BSA). The results showed that extensive sample loss (as much as 62%) was encountered during the SCX cleaning step. We then applied the RapiGest method in combination with two-dimensional LC MS/MS to characterize the E. coli membrane proteome. In total, 1626 unique proteins (5799 unique peptides) were identified with a peptide false discovery rate of 2.4%. About 60% of the identified proteins with known cellular locations were found to be membrane proteins. Among them, about 75% were integral membrane proteins. This work represents one of the most comprehensive profiles of E. coli membrane proteome generated by a proteomic technique.
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Canada.
authors.forename: Fang
authors.initials: F
authors.lastname: Wu
authors.forename: Difei
authors.initials: D
authors.lastname: Sun
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Gong
authors.forename: Liang
authors.initials: L
authors.lastname: Li
mesh_terms: D000143:Acids / Q000737:chemistry*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002411:Cation Exchange Resins / Q000737:chemistry; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D000143:Acids; D002411:Cation Exchange Resins; D008565:Membrane Proteins; D013501:Surface-Active Agents; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate