Zheng et al. BMC Genomics 2011, 12:40 http://www.biomedcentral.com/1471-2164/12/40 Page 6 of 11 Figure 4 The co-localization of mmpL8 gene. The mmpL8 gene was linked to genes involved in polyketide biosynthesis (pks2 gene) and lipid metabolism (papA1 and fadD23 genes). suggests a similar role of these proteins in complex lipid transport in mycobacteria [24]. Sequence analysis reveals MmpL8 protein comprises 1089 amino acid residues and is predicted to contain 12 membrane-spanning alpha helices. The PapA1 protein matched with four unique peptides and was a suspected membrane-asso- ciated acyltransferase, suggesting its involvement in the esterification of trehalose with methyl-branched fatty acids during the biosynthesis of polyacyltrehalose and SL-1 [25]. Additionally, FadD23 can combine with the protein encoded by pks gene in various ways to form complex hybrid metabolites [26]. This is also the case with MmpL11, which is closely related to BCG_0241c and BCG_0238c (identified in band VII). In 2-D gel, three proteins were unambiguous identified (spot 33, 34 and 35, respectively). These encoding genes occur within the same apparent gene cluster, which is conserved in all of the mycobacterial genomes sequenced to date and found to play a possible fundamental role in mycobacterial pathogenicity [27]. Since proteins MmpL8 and MmpL11 may be involved in the transportation of molecules that function in host- pathogen interactions, inhibitors of MmpL8 or MmpL11 may provide novel drug targets in the future. ATP synthase complex Gel band II comprised 11 proteins, as measured by LC MS/MS, including four IMPs. All members of ATP synthase complex were identified from this band. Reso- lution of the band on the 2-D gel revealed eight subu- nits of ATP synthase were obtained. In detail, subunit a (AtpB, spot 13), which contained five transmembrane helices, matched with two unique peptides. Subunit b (AtpF, spot 15), which embedded in the membrane and contained a long helical domain extending into the cyto- plasm, was in contact with subunit a, and subunit c (AtpE, spot 17) with an apparent molecular mass of about 8 kDa, which contained two hairpin-like TMHs, was connected by a central sequence that was exposed to the cytoplasm [28]. Subunit b (AtpD, spot 8), which constituted part of the a3b3 hexamer, contained 19 peptides and the sequence coverage was 46.5%. Addi- tionally, subunits a (AtpA, spot 7) and g (AtpG, spot 12) consisted of 14 and nine peptides, respectively (see Table 1 for details). Subunits δ (AtpH, spot 9) and ε (AtpC, spot 16) contained nine and five unique peptides, respectively. In our work, although the stoichiometry of the a3b3g complex could not be determined by MS/MS measurements, the numbers of the identified peptides represented, at least partially, the relative abundance of these subunits (14, 19 and nine unique peptides, respec- tively). In fact, using the procedure proposed by Abresch et al., we were able to estimate the molecular ratios of the subunits [29]. It is a common feature of BN PAGE that minor frac- tions of multiprotein complexes precipitate during the electrophoretic run. In fact, spot 6 on 2-D gel (Figure 3) was identified as putative lipoprotein LpqW. Analysis of lipoprotein LpqW with bioinformatic softwares pre- dicted a GRAVY score of 0.027 and no transmembrane helices. Interestingly, STRING 8.3 database analysis pre- dicted that the LpqW protein is a functional partner of integral membrane arabinosyltransferase A (EmbA, iden- tified in band IV) (confidence score > 0.6). It seemed that the LpqW protein was a cytoplasmic protein that interacted with the Emb protein complex (identified in band IV). Additionally, the putative esterase (LipO, spot 10) migrated as “free” proteins because of no attachment to other protein subunits using STRING database analy- sis. Spot 11 was identified as chaperone protein (DnaJ2), which was a subunit of heat shock protein family (iden- tified in band VI). Spot 14, aligned with other proteins in band II, was observed but no identification was obtained by MS analysis. Phenolpthiocerol synthase complex In band III on BN gel, we obtained two members of phenolpthiocerol synthase (PPS) protein family, which was encoded by a large operon [30]. In 2-D gel spot 18 and 19 were identified as PpsC and PpsA, and they matched with eight and 12 unique peptides, respectively. A previous study demonstrated that PpsC was involved in a late step in the biosynthesis of phthiocerol and phe- nolphthiocerol and disruption of PpsC in M. bovis BCG led to be an inability to produce mycoside B and phthio- cerol dimycocerosates (PDIMs) [31]. Practically, since the proteins encoded by pps genes are unique to patho- genic mycobacteria, they may be used as targets in the high throughput screening of desperately needed anti- mycobacterial drugs. Additionally, database search after MS/MS analysis of pigmented spot 20 did not yield any result. Emb protein complex BN gel band IV was subjected to in-gel digestion and analyzed by LC-MS/MS. Three proteins encoded by emb genes, EmbA, EmbB and EmbC, were identified in this band. In 2-D gel, three spots (spot 21, 22 and 23) were identified as arabinosyltransferases (EmbB, EmbC and EmbA, respectively). On the basis of sequence ana- lysis, these proteins are IMPs with 12 TMHs (excluding the possible signal peptide), and match with four, seven