Journal of Chromatography A, 1122 (2006) 35–46 Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis Henrik Everberg, Thom Leiding, Anna Schi¨ oth, Folke Tjerneld, Niklas Gustavsson ∗ Department of Biochemistry, Center for Chemistry and Chemical engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden Received 30 January 2006; received in revised form 6 April 2006; accepted 11 April 2006 Available online 6 May 2006 Abstract It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies. © 2006 Elsevier B.V. All rights reserved. Keywords: Detergents; Solubilization; Enrichment; Membrane proteins; Aqueous two-phase systems; Proteomics 1. Introduction A membrane proteome contains both integral membrane pro- teins, which span the lipid bilayer one or several times, and peripheral proteins associated with the integral membrane pro- teins or the membrane surface by covalent or non-covalent inter- actions. Two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) are traditionally employed for separation and identification of soluble proteins. However, membrane pro- teomics has been hampered due to the poor solubility of integral membrane proteins in the first dimension iso-electric focusing step of 2DE [1,2]. Proteomic strategies currently used for suc- cessful identification of membrane proteins are mainly based on high-resolution separation of peptides by liquid chromatography in combination with tandem mass spectrometry (LC–MS/MS) [3–5]. Proteins are subjected to enzymatic proteolysis in solu- ∗ Corresponding author. Tel.: +46 46 2228115; fax: +46 46 2224116. E-mail address: niklas.gustavsson@biochemistry.lu.se (N. Gustavsson). tion generally resulting in very complex peptide mixtures, and multi-dimensional separation by LC is often required to reduce sample complexity enough for protein identification by MS/MS [6]. Although this so-called shotgun proteomic approach has been successful in identifying large numbers of membrane pro- teins, a major drawback derived from the peptide-based nature of the approach is that protein-specific information, such as size, iso-electric point, isoforms and post-translational mod- ifications, is lost during the proteolysis step [7]. Thus, data derived from the peptide level of a shotgun experiment is in many cases difficult to interpret to yield information on the protein level, and even more on the level of protein–protein interactions such as for multi-subunit membrane protein com- plexes and alternative methods are thus needed. Conventional one-dimensional SDS-PAGE (1DE) is compatible with mem- brane protein separation although the resolving power is rela- tively low, compared to 2DE. Therefore, reduction of sample complexity by pre-fractionation is crucial prior to 1DE sep- aration. We have previously reported on the use of aqueous two-phase partitioning and ion-exchange chromatography for 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.04.020