Zheng et al. BMC Genomics 2011, 12:40 http://www.biomedcentral.com/1471-2164/12/40 protein complexes were found, including ATP synthase, NAD(P) transhydrogenase, ubiquinone oxidoreductase and ubiquinol-cytochrome C reductase. In detail, the soluble protein encoded by the pntAa gene has been shown to be associated with two other proteins encoded by pntAb and pntB genes, forming NAD(P) transhydro- genases complex on the membrane. Additionally, six subunits of the ATP synthase complex, five subunits of the ubiquinone oxidoreductase complex and three subu- nits of the cytochrome bc1 complex were also identified, respectively. In the present study, to confirm the protein composition of each complex, nine bands which cut out from 1-D BN gel were resolved into their individual subunits by dena- turing SDS PAGE. The subunits of each complex were ordered according to their molecular weight in vertical rows. Figure 3 showed a 2-D PAGE map of protein com- plexes identified in BN gel bands and 44 spots were recog- nizable on the map. In total, 40 distinct proteins were identified by two or more peptide hits using the Mascot algorithms. A complete list of all identified proteins is pro- vided in Table 1 and Additional file 1. On average, the identified proteins matched with more than six unique peptides. Of these proteins, 12 proteins had at least one transmembrane helix (TMH) predicted by TMHMM 2.0 (excluding the possible signal sequences predicted using SignalP). The average GRAVY value, which is used to eval- uate the hydrophilicity and hydrophobicity of a protein along with its amino acid sequence, was 0.33 for integral membrane proteins (IMPs) [23]. Based on the Pasteur Institute functional classification tree, the identified pro- teins were distributed across several functional groups (see Table 1 for details). Most of the identified proteins were involved in intermediary metabolism and respiration (Functional category 7, 27.5%). Additionally, seven pro- teins (17.5%) were required for lipid metabolism (Func- tional category 1), information pathways (Functional category 2) and cell wall and cell processes (Functional category 3), respectively. It was interesting that some pro- teins with high hydrophobicity involved in complexes were identified in our study, for example, EmbC, EmbA and EmbB, which contained 12 TMHs. Therefore, our method may offer an effective alternative strategy to iden- tify proteins with high hydrophobicity or multiple TMHs in complex. To investigate the potential protein com- plexes, the molecular composition and function of the proteins were analyzed as discussed below. Analysis of potential membrane and membrane- associated protein complexes Lipid metabolism complex In BN gel, one band migrated sharply in the upper part of the gradient gel. In this band, we identified MmpL8 and the polyketide synthase Pks2, along with the Page 4 of 11 Figure 3 2-D SDS PAGE map of multiprotein complexes from M. bovis BCG membrane fractions. Nine BN bands were loaded on top of a 12% separating gel and separated by SDS PAGE. 2-D gel was stained with colloidal Coomassie blue and left lane was PageRuler Prestained Protein Ladder (Fermentas). Roman numerals on top of the map indicated BN gel bands. The spots were excised and identified by MS/MS and the proteins were shown in Table 1. proteins PapA1, FadD23 and BCG_3890c. In 2-D gel this BN gel band was resolved into five proteins (spot 1-5). Spot number 1 migrated with an apparent molecu- lar mass of about 230 kDa and was identified as polyke- tide synthase. Spot 2 was identified as integral membrane transport protein (MmpL 8). In addition, a fatty-acid-CoA ligase (FadD23, spot 3), a polyketide synthase associated protein (PapA1, spot 4) and a puta- tive transposase (BCG_3890c, spot 5) were also found in 2-D gel. Genome analysis reveals that the mmpL8 gene is positioned 8 kbp downstream from the pks2 gene, and the papA1 gene is also linked to the pks2 gene (see Figure 4 for details). The co-localization of the mmpL gene with genes involved in polyketide biosynthesis (pks gene) and lipid metabolism (papA and fadD genes),