Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins.
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.
Everberg
Henrik
H
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
Sivars
Ulf
U
Emanuelsson
Cecilia
C
Persson
Cecilia
C
Englund
Ann-Kristin
AK
Haneskog
Lars
L
Lipniunas
Peter
P
Jörntén-Karlsson
Magnus
M
Tjerneld
Folke
F
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
D003902:Detergents; D008565:Membrane Proteins; D011108:Polymers