Zheng et al. BMC Genomics 2011, 12:40 http://www.biomedcentral.com/1471-2164/12/40 Background Tuberculosis (TB) is an infectious bacterial disease, caused primarily by Mycobacterium tuberculosis [1]. One third of the world’s population is currently infected with TB bacillus. In many countries, immunization of infants with bacillus Calmette-Guérin vaccine (BCG) protects against TB meningitis and other severe forms of TB in children less than five years of age http://www. who.int/en/. However, BCG vaccination is not recom- mended for adults because the protection provided is variable [2]. The threat to human health presented by TB worldwide is increased by the emergence of multi- drug-resistant strains and co-infection with human immunodeficiency virus [3]. Therefore, it is crucial to develop novel strategies to improve the BCG vaccine or to develop effective drugs to reduce the socioeconomic and health burden associated with TB. Membrane proteins play important roles in various cel- lular processes, including cell adhesion, cell metabolism, ion transport and signal transduction [4]. Membrane pro- teins are represented by around 30% of the genome and constitute approximately 70% of all pharmaceutical drug targets [5]. Traditional one-dimensional electrophoresis (1-DE) and two-dimensional electrophoresis (2-DE) coupled with MS are commonly used to study membrane proteome [4]. However, solubility and low abundance issues of membrane proteins remain to be the main chal- lenges in gel electrophoresis [6]. Large-scale identifica- tion of M. tuberculosis membrane proteins came forth since 2002 [7-10]. In 2007, Mattow et al. carried out membrane subproteomic analysis on M. bovis BCG Copenhagen by 1-D SDS polyacrylamide gel electrophor- esis (PAGE) and 2-DE and obtained 125 unique proteins including 54 proteins harboring 1-14 predicted TMHs [11]. In 2008, Målen et al. identified 351 proteins by a combination of both gel-based and gel-free protein and peptide fractionation methods, including 103 integral membrane proteins with at least one predicted trans- membrane region [12]. Recently, they also separated hydrophobic membrane and membrane-associated pro- teins directly from sonicated M. tuberculosis H37Rv using SDS PAGE and liquid chromatography tandem mass spectrometry (LC-MS/MS) [13]. In general, proteins rarely function completely inde- pendently, which makes identification of protein-protein interactions crucial to our understanding of numerous cellular processes [14]. A range of proteomic approaches have been adopted to analyze diverse protein interac- tions [15]. Blue native (BN) PAGE, a specialized type of native electrophoresis, is widely applied to investigate protein-protein interactions, especially for membrane proteins [16]. BN PAGE was first described in 1991 for the separation of membrane protein complexes from the respiratory chain of human mitochondria [17]. Previous Page 2 of 11 studies have demonstrated that multiprotein membrane complexes can be resolved into individual blue bands in 1-D by BN PAGE, and the protein subunits of each complex can then be separated by various types of gel electrophoresis in 2-D, for example, SDS PAGE, doubled SDS PAGE (dSDS PAGE) or IEF/SDS 3-D PAGE [18]. However, identification of the stained protein spots in 2-D PAGE by MS analysis is tedious and time-consum- ing, despite recent advances in automation [19]. Further- more, artifactual methionine oxidation that occurrs during SDS PAGE cannot be entirely prevented, which is a problem when attempting to identify physiological oxidative modifications of complexes [20]. In 2005, Babusiak et al. presented a novel native 2-D separation technique that combined native electrophoresis with native LC and could be applied to the separation and identification of intact heme-binding protein complexes in murine erythroleukemia cells [21]. The feasibility of this approach was also illustrated by Fandino et al. who combined BN PAGE and non-gel shotgun methods [19]. Wessels et al. extended this method to analyze full length of 1-D BN gel lanes and identified potentially interacting proteins by protein correlation profiling (PCP) [14]. Since these methods are fully compatible with MS/MS protein identification, they could be widely applied in the study of membrane protein complexes. Studies on a variety of membrane protein complexes have been reported, however, little is known about mycobacterial membrane complexes. Here we investi- gated M. bovis var BCG NCTC 5692, an attenuated derivative of M. bovis, by an approach coupling BN PAGE to LC-MS/MS to analyze potential protein- protein interactions in membrane fractions. All proteins identified in 1-D BN bands were confirmed using 2-D gel. We have obtained a profile of the M. bovis BCG potential membrane and membrane-associated protein complexes, and these results may provide some insights into BCG protein interaction studies. Results and discussion Sample pre-treatments for BN PAGE The aim of this work is to isolate and characterize native and structurally intact membrane protein com- plexes directly from BCG membrane fractions. The pro- tein complex stability during the sample preparation and separation is an extremely important factor in effec- tive identification. It has been demonstrated that some detergents allow for effective solubilization of proteins in oligomeric state, maintenance of enzyme activity and sharp resolution in electrophoresis [22]. Mild non-ionic detergents, Triton X-100 and n-dodecyl-b-D-maltoside (DDM), were chosen to solubilize membrane fractions and the effect of different concentrations of detergents was tested experimentally. Briefly, two samples of the