title: Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins.
abstract: Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (1-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes.
authors.affiliation: Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Everberg
authors.forename: Ulf
authors.initials: U
authors.lastname: Sivars
authors.forename: Cecilia
authors.initials: C
authors.lastname: Emanuelsson
authors.forename: Cecilia
authors.initials: C
authors.lastname: Persson
authors.forename: Ann-Kristin
authors.initials: AK
authors.lastname: Englund
authors.forename: Lars
authors.initials: L
authors.lastname: Haneskog
authors.forename: Peter
authors.initials: P
authors.lastname: Lipniunas
authors.forename: Magnus
authors.initials: M
authors.lastname: Jörntén-Karlsson
authors.forename: Folke
authors.initials: F
authors.lastname: Tjerneld
mesh_terms: D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011108:Polymers / Q000737:chemistry*; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D011108:Polymers