The gel spots were then dehydrated in selleck screening library acetonitrile for 30′ and dried in a speed vac for 10′. Thirty microliters of 50 mM ammonium bicarbonate containing 0.3 μg of trypsin (Sigma-Aldrich, St Louis, MO) were added to each sample, and samples were incubated at 37°C for 16 hours. Digested peptides were extracted from gel spots by two washes of 50% acetonitrile/0.1% trifluoroacetic acid, and purified with Ziptips

(Millipore, Billerica, MA). Purified peptides were eluted from Ziptips with 50% acetonitrile/0.05% trifluoroacetic acid with 10 mg/ml alpha-cyano-4-hydroxycinnamic acid, and spotted on a sample plate to obtain mass spectra using an Axima CFR Plus MALDI-ToF mass spectrometer (Shimadzu Biotech, Columbia, MD). Each spectrum was calibrated externally using the ProteoMass peptide MALDI-MS calibration kit EX 527 research buy (Sigma-Aldrich, St Louis, MO). Peptide fingerprints obtained for each sample

were used to search the databases at NCBI and SWISS-PROT using MASCOT search engine http://​www.​Matrixscience.​com. Search parameters used were variable carbamidomethyl and propionamide modifications of cysteines and oxidation of methionines. A peptide tolerance window of 0.5 daltons was used for all searches. Once an identification was made with a statistically significant score, data were accepted when the peptide coverage of the protein was at least 20%, and the molecular weight and isoelectric point of the protein matched those observed on the 2D gel electrophoresis. Acknowledgements We thank Drs. Stuart Linn and Hiroshi Nikaido for insightful JNK-IN-8 price discussions. This work was supported by USDA CALR-2005-01892 (to S. L.). References 1. Hoch JA: Two-Component Signal Transduction Washington, DC: American Society for Microbiology Press 1995. 2. Nixon BT, Ronson CW, Ausubel FM: Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation SPTLC1 regulatory genes ntrB and ntrC. Proc Natl Acad Sci USA 1986, 83:7850–7854.CrossRefPubMed 3. Iuchi S, Weiner L: Cellular and molecular physiology of Escherichia coli in the adaptation to aerobic environments. J Biochem (Tokyo) 1996, 120:1055–1063. 4. Bauer

CE, Elsen S, Bird TH: Mechanisms for redox control of gene expression. Annual Review of Microbiology 1999, 53:495–523.CrossRefPubMed 5. Hidalgo E, Ding H, Demple B: Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem Sci 1997, 22:207–210.CrossRefPubMed 6. Demple B: Study of redox-regulated transcription factors in prokaryotes. Methods 1997, 11:267–278.CrossRefPubMed 7. Ding H, Demple B: Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein. Proc Natl Acad Sci USA 1996, 93:9449–9453.CrossRefPubMed 8. Nunoshiba T, Hidalgo E, Amabile Cuevas CF, Demple B: Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene.