Coupling of Native Liquid Phase Isoelectrofocusing and Blue Native Polyacrylamide Gel Electrophoresis: A Potent Tool for Native Membrane Multiprotein Complex Separation Gian Maria D’Amici, Anna Maria Timperio, and Lello Zolla* Department of Environmental Sciences, Tuscia University, Viterbo, Italy Received September 21, 2007
Abstract: In this study, a new 3D native electrophoretic protocol is proposed for an exhaustive separation and identification of membrane proteins. It is based on native liquid phase isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second dimension, where both the pI and the molecular masses of protein complexes (2D N-LP-IEF-BN) were used to separate them in their native form. Finally, each single component can be resolved using denaturing electrophoresis (3D N-LP-IEF-BN-SDS-PAGE). The thylakoid membrane of spinach which contains four big protein complexes was chosen as a model for setting up analytical methods suitable for any membrane proteins. The pIbased MicroRotofor has a number of advantages over BNPAGE: it does not require the addition of any chemicals, and separation of complexes is based on the protein’s real physicochemical properties which inevitably change when dye is added. Results were more easily reproduced than with BN, and the pI of each native complex was also determined. Although some fractions still contained comigrating complexes after MicroRotofor, these were subsequently separated by BN for further analysis. Thus, highly hydrophobic complexes, such as ATP-synthetas and Cyt b6/f, were separated in native form as were various complexes of LHCII trimers, which have different pI but similar molecular masses. SDS-PAGE revealed almost all the subunits from the four photosynthetic complexes, indicating that by using 3D N-LP-IEF-BN-SDSPAGE it is possible to achieve a greater degree of component identification than with 2D BN-SDS-PAGE. Keywords: proteomics • blue native PAGE • native liquid phase IEF • nondenaturing electrophoresis • membrane proteins • thylakoid membrane proteins
Introduction Most proteome analyses carried out to date have either been concerned with the location of proteins within cells or have involved a differential analysis of expression in response to * Corresponding author: Prof. Dr. Lello Zolla, Tuscia University, Largo dell′ Università snc, 01100, Viterbo, Italy. Phone: 0039 0761 357 100. Fax: 0039 0761 357 630. E-mail:
[email protected].
1326 Journal of Proteome Research 2008, 7, 1326–1340 Published on Web 02/05/2008
development, environment, or disease. As such, they have not been able to provide information about the way these proteins interact, which is a very important aspect of understanding the functions of these proteome members. The functional proteomics of membrane proteins is an important tool for understanding protein networks in biological membranes, and this is particularly challenging since they tend to self-associate into noncovalent multimers due to their hydrophobic nature. Similarly, most cellular processes require the action of several enzymes, each often containing multiple subunits associated with each other, forming temporary or stable larger protein complexes. Thus, knowledge of the composition and/or structure of these protein complexes, as well as relevant protein interactions, will result in a much deeper understanding of metabolic pathways and cellular processes than protein identities alone are able to deliver, especially if the complexes can be separated in the native form. There are many ways to investigate hydrophobic complexes and protein interactions, each with their own advantages and drawbacks.1 However, with few exceptions, most approaches focus on a single protein and determine its binding partners. Moreover, these approaches are not designed to provide a general overview of protein–protein interactions in a complex proteome of choice by a single experiment. This emphasizes the need for complementary ways to analyze in vivo protein interactions where complexes can be separated in native form. Electrophoresis remains an elective method for protein separation and identification, although some limitations need to be improved. Most studies have used a variety of approaches to separate proteins, but isoelectric focusing (IEF) followed by SDS-PAGE (typically termed a 2D gel) is the most common technique undertaken to resolve large numbers of soluble and peripheral membrane proteins.2 However, it is notoriously difficult to achieve high-quality resolution of hydrophobic membrane proteins using 2D IEF-SDS-PAGE. In Escherichia coli, for example, it was estimated that around 30% of proteins with positive grand average hydropathy (GRAVY) scores did not produce spots on 2D-IEF-SDS-PAGE gels.3 The poor separation of hydrophobic proteins in 2D electrophoresis comes from the aggregation, oxidation, and precipitation which proteins undergo during the first IEF dimension.4 It goes without saying that this method is not suitable for the isolation of native protein complexes. In this regard, Schägger et al.5 developed a novel experimental strategy where through the combination of mild detergents and the dye Coomassie blue, substituting for the highly denaturating detergent sodium dodecyl sulfate 10.1021/pr700613r CCC: $40.75
2008 American Chemical Society
2D N-LP-IEF-BN-PAGE: A Nondenaturing Electrophoresis System (SDS), it was possible for the first time to separate intact mitochondrial respiratory multiprotein complexes (MPCs) by electrophoresis. This technique has been named BN-PAGE. Protein complexes migrate to the anode and are separated by the decreasing pore size of the polyacrylamide gradient gel according to the particle size (apparent molecular mass) which can separate protein complexes in their native form while retaining their individual subunit proteins.6 This excellent gel system can resolve membrane protein complexes with greater resolution than gel filtration or sucrose density gradient. It is suitable for separating protein complexes in the range of 10 KDa to 10 MDa, allowing the separation of extremely hydrophobic protein sets for analysis while also providing information on their native interactions. However, if the first native dimension is combined with a second dimensional SDS-PAGE, it is also possible to dissociate the individual subunit proteins of complexes. Native gel image analysis can be a powerful instrument for semiquantitative estimation of the variation found in complexes formed under stress conditions.7 This makes BN-PAGE the preferred method for the analysis of hydrophobic proteomes as it does not seem to suffer from the problems encountered in 2D electrophoresis with IEF.3,4 There is now a growing number of publications where this method has been employed for the investigation of other hydrophobic and hydrophilic high molecular weight protein complexes in different organisms. Unfortunately, complexes displaying close molecular masses cannot be separated by this technique, and weak or transient interactions between protein complexes can reduce the quality of separation. Single proteins or complexes of a molecular weight
