5 days and 4 days post inoculation, respectively. The VS-4718 nmr expression of bacterial DnaK was used as the internal control. Protein samples were reacted with antibodies against the FLAG sequence (top panel) and DnaK (low panel). Each lane was loaded with material from

5 × 107 CFU bacteria. (C-D). Level of tagged proteins from the bacterial CA4P ic50 strains recovered from the macrophages and spleens of infected mice as determined in (A) and (B). The values, which are the means of triplicate experiments, represent the relative percentage of the levels of the tagged proteins in the bacteria recovered from macrophages (C) at 5 hours postinfection and from the spleen at 5 days postinoculation (D), as compared to those in the bacteria recovered from macrophages at 0.2 hours postinfection and from spleen at

0.5 days post inoculation, respectively. In cultured macrophages, SipA, SipC, and SopB were all expressed at the early phase (e.g. 0.2 h) of infections. However, by 5 hr post infection, the levels of the three SPI-1 proteins diverged, with the SipC level increased, the SopB level decreased while SipA level remained unchanged (Figure 6A and 6C). To determine the relative abundance of these proteins in the spleen during systemic infection, BALB/c mice were infected intraperitoneally. Salmonella was recovered from the spleen at different time points postinfection, and selleck chemicals llc the expression levels of the tagged proteins were determined. Similar to the results of macrophage infection, all three proteins were

detected during the early stage of infection (i.e. 0.5 days). However, at a later stage of systemic infection (i.e. 5 days), the level of SipC increased and the level of SopB decreased while the level of SipA remained unchanged (Figure 6B and 6D). These results correlated with those observed in the proteomic analyses and in the macrophage experiments. Furthermore, these data strongly suggest that different SPI-1 factors are specifically expressed at late stage of Salmonella infection, and highlight a possible role of SipC in late phase of macrophage and in vivo infections of Salmonella. Discussion Stable isotope labeling procedure coupled with MS-based analysis for quantitative very proteomic study of bacterial protein expression In the postgenomic era, new methodologies are needed that can quantitatively, globally, and accurately measure protein expression in cells and tissues [37]. In this study, we have modified the SILAC method to develop a stable isotope labeling procedure coupled with MS analysis to carry out quantitative proteomic analysis of Salmonella. As a “”proof of principle”" pilot study, a total of 103 SE2472 proteins were monitored for their expression profiles upon exposure to H2O2. At least seventy six proteins have been found to be modulated in the presence of H2O2.