Probability-based scoring method with MASCOT database search engine (Matrix Science, Boston, MA) was used to identify each protein, based on the likelihood of search results being a random match. We used the following parameters for our protein identification: Database: NCBINR, MASCOT value cut off: greater than 62 (p < Vemurafenib 0.05), Taxonomy: Salmonella, Missed cleavage: 1, Peptide Tolerance: +/- 0.75 Da, Variable modification:
none, Fixed modification: none, Enzyme: Trypsin, Mass Values: Monoisotopic. Quantitative analysis Tryptic peak data from MASCOT database searches was tabulated and elemental composition of each peptide fragment was determined using an in-house data analysis software. The process was further automated using a custom VBScript written for Microsoft Excel, which was designed to calculate predicted 15N peak location based on the primary amino acid sequence of tryptic peptide fragments. 14N/15N mixture MS spectrum was used to obtain GSK461364 mouse peak intensity ratio between labeled (15N) and unlabeled (14N) samples
to give relative quantification data. An average of 10 peaks was used to calculate the mean intensity ratios and the error percentage of each protein spot. Blebbistatin molecular weight Significant outliers were manually removed from the data set to prevent them from affecting the results (less than 2%). To further increase the accuracy of our results, experiments were preformed three times, and the results were the average from the triplicate experiments. Only those proteins that were detected and identified with high confidence in all three independent experiments are listed in Table 1 and Table 2. Growth and survival analysis of Salmonella Strains SipA(HF), SipC(HF) and SopB(HF) are derivatives of the wild type Salmonella enterica serovar Enteritidis strain SE2472 Amylase with a FLAG tag inserted in-frame at the C-terminus of each corresponding protein and have been described previously . Growth analysis of bacteria in LB or LB-like broth was carried out by first inoculating a single colony
in 2 ml of either normal (14N) or 15N-labeled media and culturing at 37°C with shaking at 225 RPM overnight (about 16 hours) . Thirty microliters of the overnight culture were then inoculated into 3 ml fresh normal or 15N-labeled media or LB broth and cultured at 37°C with shaking at 225 RPM. At 0, 2, 4, and 6 hours after inoculation, 100 μl of bacterial culture were collected to determine their colony forming unit (CFU)/ml by plating. Salmonella grew in normal (14N) or 15N-labeled media as well as in LB broth (data not shown). To study the survival of Salmonella after exposure to H2O2, 20 μl of the overnight culture grown in normal (14N) or 15N-labeled media, or LB broth were added to 2 ml of fresh normal (14N) or 15N-labeled media, or LB broth containing 5 mM H2O2.