Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels An extensive proteomic approach relies on the possibility to visualize and analyze vari- ous types of proteins, including hydrophobic proteins which are rarely detectable on two-dimensional electrophoresis (2-DE) gels. In this study, two methods were em- ployed for the purification of hydrophobic proteins from Arabidopsis thaliana leaf plas- ma membrane (PM) model plants, prior to analysis on 2-DE immobilized pH gradient (IPG) gels. Solubilization efficiency of two detergents, (3-[(3-cholomidopropyl)-1-pro- panesulfonic acid (CHAPS) and C8é, were tested for the recovery of hydrophobic pro- teins. An immunological approach was used to determine the efficiency of the above methods. Fractionation of proteins by Triton X-114 combined with solubilization with CHAPS resulted in the inability to detect hydrophobic proteins on 2-DE gels. The use of C8é for protein solubilization did not improve this result. On the contrary, after treat- ment of membranes with alkaline buffer, the solubilization of PM proteins with deter- gent C8é permitted the recovery of such proteins on 2-DE gels. The combination of membrane washing and the use of zwitterionic detergent resulted in the resolution of several integral proteins and the disappearance of peripheral proteins. In the resolution of expressed genome proteins, both large pH gradients in the first dimension and vari- ous acrylamide concentrations in the second dimension must be used. Notwithstand- ing, it is important to combine various sample treatments and different detergents in or- der to resolve soluble and hydrophobic proteins. Keywords: Arabidopsis thaliana / Hydrophobic protein / Plasma membrane / Proteome / Two- dimensional electrophoresis EL 3392 VØronique Santoni 1 Thierry Rabilloud 2 Patrick Doumas 1 David RouquiØ 1 Monique Mansion 1 Sylvie Kieffer 3 JØrôme Garin 3 Michel Rossignol 1 1 Biochimie et Physiologie MolØculaire des Plantes, INRA/ENSA-M/CNRS URA 2133, Montpellier, France 2 BioØnergØtique Cellulaire et Pathologique, DBMS, CEA- Grenoble, France 3 Laboratoire de Chimie des ProtØines, CEA-Grenoble, Grenoble, France 1 Introduction Proteome research provides a powerful tool to comple- ment other approaches currently used in molecular biolo- gy through analysis of the actively translated portion of the genome. The use of IPG (immobilized pH gradient) has greatly improved the feasibility of such approaches and interlaboratory reproducibility [1]. Currently most of the proteomes published have been achieved across pH range 4±8 and using only one given concentration of acrylamide in the second dimension. However, analysis of the expressed genome needs to be as complete as possible and should not be limited to proteins with a par- ticular pI and molecular weight [2]. Recently, large immo- bilized pH gradients and different acrylamide concentra- tions were used [3, 4] in order to recover the maximum amount of proteins possible. An extensive proteomic ap- proach also relies on the possibility to visualize specific types of proteins (i.e., hydrophobic proteins, glycosylated or membrane-anchored proteins). However, such hydro- phobic proteins are rarely detectable on 2-DE gels [5, 6]. In fact, a recent study on the characterization of the plas- ma membrane (PM) from the plant model Arabidopsis thaliana demonstrated that approximately 80% of PM- specific proteins detected in 2-DE gels corresponded nearly exclusively to extrinsic proteins [7]. A combination of recently synthetized new detergents [8] and thiourea was proposed to improve the recovery of hydrophobic proteins on 2-DE gels. In the case of the plant PM, two of the most abundant hydrophobic proteins (water channels and H + -ATPase) were effectively detected after solubili- zation with C8é [8]. In this study, we further focused on hydrophobic proteins from Arabidopsis thaliana PM and compared the efficien- cy of various treatments designed to enrich the sample in hydrophobic proteins. Using an immunological approach together with protein identification to assess the presence of hydrophobic proteins, a specific procedure was de- rived. This procedure is compatible with the use of C8é and enables the selectively improved recovery of hydro- phobic proteins. Correspondence: Dr. VØronique Santoni, Biochimie et Physiolo- gie MolØculaire des Plantes, INRA/ENSA-M/CNRS URA 2133, place Viala, 34060 Montpellier cedex 1, France E-mail: santoniv@ensam.inra.fr Fax: +33-4-67-52-57-37 Abbreviation: PM, plasma membrane Electrophoresis 1999, 20, 705±711 705  WILEY-VCH Verlag GmbH, 69451 Weinheim, 1999 0173-0835/99/0405-0705 $17.50+.50/0 Proteomics and 2-DE