Zheng et al. BMC Genomics 2011, 12:40 http://www.biomedcentral.com/1471-2164/12/40 and three unique peptides, respectively. Functional ana- lysis showed that EmbC is involved in the synthesis of lipoarabinomannan, and EmbA and EmbB are responsi- ble for the polymerization of arabinose into the arabinan of AG [32]. They have been proposed to be the main targets of ethambutol (EMB). Disruption of these proteins would inhibit the formation of the mycolyl- arabinogalactan-peptidoglycan complex and may lead to increased permeability of the cell wall and drug uptake [33]. Therefore, they may serve as targets for the devel- opment of therapeutic approaches against TB. In this band, a putative membrane alanine rich pro- tein was obtained (BCG_2759c, spot 24). Analysis of the primary sequence of the protein predicted two trans- membrane helices and STRING database analysis pre- dicted that the protein BCG_2759c was no interaction among those proteins identified in our study. In this way, we were able to conclude that the protein BCG_2759c was a cytoplasmic membrane protein and a “free” protein because of no clear attachment to other protein identified in 2-D gel. In addition, spot 25 was identified as subunit a of ATP synthase complex (identi- fied in band II). Polyketide synthases Two polyketide synthases (PKSs), Pks7 and Pks8, were identified in BN band V. Genome analysis reveals that pks7 and pks8 gene are located in a cluster of pks genes. The proteins encoded by these genes are large multi- functional enzymes and belong to the same PKS family required for PDIM synthesis in mycobacteria [34]. In 2-D gel, spot 26 migrated with an apparent molecular mass of great than 200 kDa was identified as Pks7, and the protein matched with 12 unique peptides. It has been reported that Pks7 plays an important role in viru- lence during the persistent phase of infection [35]. Addi- tionally, Pks8 (spot 27) matched with six peptides, and has been implicated in the biosynthesis of monomethyl- branched unsaturated fatty acids [36]. Heat shock protein family Heat shock proteins (Hsps) are involved in a highly con- served family, and first recognized by their upregulated expression in response to host exposure to raised tem- peratures [37]. In the present study, we identified GroEL1, GroEL2, DnaK, DnaJ and FurA in gel band VI (see Table 1 for details). In 2-D gel, spots 29 and 30 were identified as the 60 kDa chaperonin 2 (GroEL 2) and 1 (GroEL 1), respectively. It has been reported that 60 kDa chaperonins are one type of molecular chaper- ones found on the surface of cells and these proteins have high affinity for lipid monolayers and bilayers and can associate with lipid membranes [38]. Accordingly, it was no great surprise to detect the presence of two 60 kDa chaperonins (GroEL1 and GroEL2) in the mem- brane fractions. Page 7 of 11 Additionally, DnaJ (spot 31) is a stress gene-encoding protein that is highly conserved among bacterial genera. Therefore, gene dnaJ is useful not only for identification of mycobacterial species but also for inferring their phy- logenetic relationships [39]. Suzanna et al. found that during the folding of some proteins, DnaK (spot 28) recognized extending polypeptide chains and cooperated with DnaJ in stabilizing this intermediate conformation [40]. In addition, spot 32 was identified as a ferric uptake regulation protein (FurA). On the basis of bioin- formatics analysis, FurA was a cytoplasmic protein and no interaction with Hsp family members. Thus, the pro- tein migrated as “free” proteins in 2-D gel. Polyribosomal proteins In general, ribosomes are ribonucleoprotein particles consisting of two subunits, which are designated as the small subunit (30S) and the large subunit (50S) in bac- teria [41]. Ribosomal proteins involved in the subunits are the major components of the polyribosome complex where mRNA is translated into protein [9]. In this study, seven ribosomal proteins were detected in band VIII, including five proteins in the large ribosomal subu- nit (L2, L5, L13, L18 and L22, see Table 1 for details) and two in the small ribosomal subunit (S4 and S5). All these proteins identified in BN gel were confirmed using 2-D SDS gel. Despite the lack of predicted TMHs, some ribosomal proteins are associated with the plasma mem- brane by IMPs and may interact transiently with the nascent membrane or secreted proteins [33]. It is impor- tant to note that some ribosomal proteins can easily dis- sociate from polyribosomal complex during their preparation and separation. Succinate dehydrogenase complex Members of succinate dehydrogenase (SDH) complex were found in band IX. SDH is a membrane-bound pro- tein complex and key enzyme participating in intermedi- ary metabolism and aerobic energy production in bacteria [42]. This complex is composed of four subu- nits SdhA/B/C/D. Here we were only able to identify two subunits: SdhA and SdhB (spot 43 and 44, respec- tively). MS analysis indicated that SdhA matched with 20 peptides, which was the highest number of unique peptide reported in our study. Intriguingly, these two subunits can exhibit SDH activity in the absence of SdhC/D, which are the membrane components [43]. In addition to the proteins discussed above, some spots were observed in 2-D gel but no identifications were found by MS analysis. For example, there were five spots presented in band VI (spot a, b, c, d and e, see Figure 3 for spots without circles) which showed exo- genous stains on 2-D denaturing gel, while no proteins were determined among these spots. The spots without identifications also presented in other bands (including band IV, V and VII, spots were not showed in Figure 3).