title: Shotgun analysis of membrane proteomes by an improved SDS-assisted sample preparation method coupled with liquid chromatography-tandem mass spectrometry.
abstract: Analysis of the membrane proteins, particularly the integral membrane proteins, is limited by the inherent membrane hydrophobicity. Sodium dodecyl sulfate (SDS) is one of the most efficient reagents used for the extraction of membrane proteins, but its presence in samples interferes with LC-MS-based proteomic analyses because it affects RP-LC separations and electrospray ionization. In this paper, we present an improved sample preparation strategy based on SDS-assisted digestion and peptide-level SDS-removal using an optimized potassium dodecyl sulfate (KDS) precipitation method (SSDP method) for shotgun analysis of the membrane proteome. This method utilizes a high concentration of SDS (1.0%) to lyse the membranes and to solubilize the hydrophobic membrane proteins, resulting in a more complete protein digestion in the diluted SDS buffer (0.1% SDS), and a high efficiency of SDS removal and peptide recovery by the optimized KDS precipitation for protein identification. The SSDP method provides evidence that proteins can be efficiently digested, and the SDS can be decreased to <0.01% allowing >95% peptide recovery. Compared to other sample preparation methods commonly used in shotgun membrane proteomics, the newly developed method not only increased the identified number of the total proteins, membrane proteins and integral membrane proteins by an average of 33.1%, 37.2% and 40.5%, respectively, but also leading to the identification of highest number of matching peptides. All the results showed that the method yielded better recovery and reliability in the identification of the proteins especially the highly hydrophobic integral membrane proteins, and thus providing a promising tool for the shotgun analysis of membrane proteome.
authors.affiliation: Key Laboratory of Tea Science of Ministry of Education and Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, PR China. ly2005306@163.com
authors.forename: Yong
authors.initials: Y
authors.lastname: Lin
authors.forename: Huajun
authors.initials: H
authors.lastname: Jiang
authors.forename: Yujun
authors.initials: Y
authors.lastname: Yan
authors.forename: Bin
authors.initials: B
authors.lastname: Peng
authors.forename: Jinhua
authors.initials: J
authors.lastname: Chen
authors.forename: Haiyan
authors.initials: H
authors.lastname: Lin
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D022781:Hepatocytes / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D011189:Potassium Chloride; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D010446:Peptide Fragments; D020543:Proteome; D012967:Sodium Dodecyl Sulfate; D011189:Potassium Chloride