SaschaRexroth JürgenM.W.Meyer zuTittingdorf FrankKrause NorbertA.Dencher HolgerSeelert Damstadt University of Technology, Department of Chemisty, Physical Biochemistry, Darmstadt, Germany Thylakoidmembraneatalteredmetabolicstate: Challengingtheforgottenrealmsoftheproteome Analysis of the membrane integral proteome is mainly dependent on the ability of protein separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique capable of efficient membrane protein separation, so far mainly applied to the mitochondrial oxidative phosphorylation machinery. Applying BN-PAGE to the thylakoid membranes after mild solubilization with digitonin we succeeded in display- ing the response of the green algae Chlamydomonas reinhardtii to altered culture con- ditions. In addition, by peptide mass fingerprinting and matrix assisted laser desorp- tion/ionization-mass spectrometry (MALDI-MS) extremely hydrophobic subunits of the photosystem complexes with 5–11 transmembrane helices were identified, which could not be accessed by in-gel digestion in previous studies. Keywords: Blue-native electrophoresis / Chlamydomonas reinhardtii / Matrix assisted laser de- sorption/ionization-mass spectrometry / Membrane protein DOI 10.1002/elps.200305543 1 Introduction Two-dimensional electrophoresis based on isoelectric focusing with immobilized pH gradients in the first dimen- sion and SDS-PAGE in the second dimension is the standard method of protein separation in proteomic approaches. However, despite recent developments [1, 2] and systematic evaluation of detergents [3], isoelectric focusing is still not the method of choice for separation of hydrophobic membrane proteins. Membrane proteins represent at least 30% of all proteins coded in the ge- nome [4]. Due to their interfacial location, membrane proteins play key roles in cellular processes like energy conversion, immunorecognition, signal transduction, and metabolite transport. For differential display, hardly de- tectable intrinsic membrane proteins are even of higher significance than high-abundant soluble proteins. Mem- brane proteins are in general underrepresented, some- times even absent in 2-D gels. Many reports of proteomic approaches with impressive number of identified proteins contain no or only a few rather hydrophilic membrane pro- teins [5, 6]. Integral membrane proteins identified in pre- sent studies are often from prepurified preparations [7, 8] or were found with specialized detection methods [9]. Membrane proteins are generally rather low abundant in eukaryotic cells when whole-cell extracts are examined. Thus, many approaches to investigate complete pro- teomes of a given cell type often fail to identify any mem- brane protein [5, 6, 10]. Due to their hydrophobic nature, membrane proteins are poorly soluble in the solvents used for IEF. They tend to precipitate either at their appli- cation position or during the IEF run close to their iso- electric point, where their solubility is decreased even more. Even the initial extraction step to solubilize mem- brane proteins from the sample may be critical and is highly dependent on detergent selection [3]. Some re- ports strengthen the view that standard chaotrope solu- tions are not appropriate for solubilization of integral membrane proteins [11, 12]. In addition, membrane pro- teins tend to have more basic isoelectric points [13], so that they could be missed on standard 2-D gels [1]. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is an electrophoresis technique capable of separating native, catalytically active membrane protein complexes [14–16]. The technique was recently reviewed by Schäg- ger [17], who introduced blue-native electrophoresis for the investigation of the inner mitochondrial membrane [18–20]. BN-PAGE employs the anionic dye Coomassie Brilliant Blue G-250, which binds noncovalently to the proteins, to transfer a negative charge to the membrane protein complexes. Since the complexes are retained in a structural intact form, hydrophilic subunits of mem- brane protein complexes are still connected to hydro- Correspondence: Sascha Rexroth, Holger Seelert, Darmstadt University of Technology, Department of Chemistry, Physical Bio- chemistry, Petersenstrasse 22, D-64287 Darmstadt, Germany E-mail: srex@gmx.de and seelert@pop.tu-darmstadt.de Fax: +49-6151-164171 Abbreviations: BN-PAGE, blue-native polyacrylamide gel elec- trophoresis; CF 0 F 1 , chloroplast ATP synthase; GRAVY , grand average of hydropathicity; LHCI, light-harvesting complex I; LHCII, light-harvesting complex II; PSI, photosystem I; PSII, photosystem II; RubisCO, ribulose-1,5-bisphosphatecarboxyl- ase/oxygenase 2814 Electrophoresis 2003, 24, 2814–2823  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/1608–2814 $17.501.50/0