title: Extraction, Enrichment, Solubilization, and Digestion Techniques for Membrane Proteomics.
abstract: The importance of membrane proteins in biological systems is indisputable; however, their amphipathic nature makes them difficult to analyze. In this study, the most popular techniques for extraction, enrichment, solubilization, and digestion are compared, resulting in an overall improved workflow for the insoluble portion of Saccharomyces cerevisiae cell lysate. Yeast cells were successfully lysed using a French press pressure cell at 20 000 psi, and resulting proteins were fractionated prior to digestion to reduce sample complexity. The proteins were best solubilized with the addition of ionic detergent sodium deoxycholate (1%) and through the application of high-frequency sonication prior to a tryptic digestion at 37 °C. Overall, the improved membrane proteomic workflow resulted in a 26% increase in membrane protein identifications for baker's yeast. In addition, more membrane protein identifications were unique to the improved protocol. When comparing membrane proteins that were identified in the improved protocol and the standard operating procedure (176 proteins), 93% of these proteins were present in greater abundance (higher intensity) when using the improved method.
authors.affiliation: Chemistry Department University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Moore
authors.affiliation: Chemistry Department University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Hess
authors.affiliation: Chemistry Department University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: James W
authors.initials: JW
authors.lastname: Jorgenson
mesh_terms: D002853:Chromatography, Liquid; D045424:Complex Mixtures / Q000737:chemistry; D003840:Deoxycholic Acid / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011312:Pressure; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility; D013010:Sonication; D014357:Trypsin / Q000737:chemistry
chemical_list: D045424:Complex Mixtures; D003902:Detergents; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D003840:Deoxycholic Acid; D014357:Trypsin
keywords: Saccharomyces cerevisiae; membrane proteins; sample preparation techniques; sodium deoxycholate; sonication