Total proteins from the 14N- and 15N- samples were extracted and quantified. A 1:1 (by weight) mixture of two samples was prepared see more and 200 μg of total proteins were separated by two-dimensional (2-D) gel electrophoresis. Visualization by silver staining revealed approximately

200 protein spots across the pI and molecular weight range of the gel, which were further investigated using quantitative proteomics (Figure 3). Figure 3 Two-dimensional gel electrophoresis of S. Enteritidis SE2472 total proteins. Approximately 200 μg of total SE2472 proteins were loaded onto a 2 D gel and visualized by the silver staining method. Analysis with matrix-assisted laser desorption/ionisation-time of AZD3965 mouse flight (MALDI-ToF) mass spectrometry was performed to map tryptic fragments from the mixture of the 14N- and 15N-(unexposed and H2O2-exposed) samples, where two sets of peptide fingerprints appear on the same spectrum (Figure 4, Table 1). We distinguished the two sets of peaks by initially using the 14N peaks

to identify the protein and amino acid contents of each peak (Figure 4 and Table 1), then using peak information to deduce the location of the 15N peaks. The ratio of the peak heights (15N/14N) was then used for relative quantification (Figures 1 and 4). Figure 4 shows an example taken from a protein sample, a tryptic peptide fragment FTGWYDVDLSEK (MW 1459.81) from S. Enteritidis phosphoglyceromutase. A peak at m/z 1473 represents the 15N-labeled population (Figure 4, upper spectrum), which does not appear in the unlabeled population. The

ratio of two peak intensities (27 and 17, respectively) represents a relative protein expression level of 0.6, or a 40% NADPH-cytochrome-c2 reductase downregulation. To further increase the accuracy of our results, each set of experiments was repeated three times. Only those proteins that were detected and identified with high confidence in all three independent experiments are listed in Table 2. Table 1 MALDI-ToF analysis and identification of SE2472 proteins. Locus Tag Description Gene Mass (KDa) pI Coverage PSLT011 Dlp (SrgA) srgA 24.74 8.58 38% STM0007 Transaldolase B talB 35.15 5.09 19% STM0012 Chaperone protein dnaK (Heat shock protein 70) dnaK 69.2 4.84 22% STM0013 Chaperone protein dnaJ dnaJ 41.31 8.41 25% STM0093 Organic solvent tolerance protein Imp 89.8 5.21 23% STM0102 L-arabinose isomerase araA 55.89 5.88 23% STM0158 Aconitate hydratase 2 acnB 82.2 5.35 29% STM0217 Elongation factor Ts tsf 33.18 5.16 41% STM0316 Aminoacyl-histidine dipeptidase pepD 52.69 5.17 15% STM0432 Phosphonoacetaldehyde hydrolase phnX 28.57 5.58 41% STM0435 Nucleotide-binding protein yajQ 18.31 5.6 52% STM0447 Trigger factor tig 48.02 4.84 23% STM0488 Adenylate kinase adk 23.49 5.