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6) in rich medium (Luria Bertani [LB] medium for P.?aeruginosa; tryptic soy broth [TSB] medium for S.?aureus) at 37��C and then treated with and without 10?mM H2O2. After 20?min, cells were collected and bacterial protein extracts were prepared by sonication. The extracts were treated for 1?hr with the IA probe for alkylating reactive cysteines in the bacterial proteomes. An isotopically labeled, protease-cleavable azido-biotin tag ( Weerapana et?al., 2010) was then attached BMS-754807 solubility dmso to the alkyne using click chemistry ( Rostovtsev et?al., 2002; Torn?e et?al., 2002). Isotopically heavy (13C and 15N) and light (12C and 14N) tags were conjugated to the ?H2O2 and?+H2O2 samples, respectively. Equal amounts of the corresponding ? and?+ H2O2 samples were mixed and subjected to?successive protease digestion BLZ945 ic50 steps with trypsin and tobacco-etch virus (TEV) protease, affording isotopically tagged, cysteine-containing peptides for mass spectrometry (MS) analysis. The light/heavy (+H2O2/?H2O2) ratio calculated for each IA-labeled cysteine in the bacterial proteomes provided a measure of its relative reactivity in?+ versus ? H2O2 samples. Oxidation of?a cysteine by H2O2 should reduce its reactivity with the IA probe, resulting in a light/heavy peptide ratio of INSRR (75.6% of all oxidation-sensitive cysteines) in P.?aeruginosa, but only 6 cysteines (5.3%) in S.?aureus ( Table 1). We define these cysteines as highly oxidation-sensitive cysteines. Previously, 18 proteins were identified as possessing oxidation-sensitive thiols after H2O2 treatment in S.?aureus through the use of a fluorescence thiol-modification assay ( Wolf et?al., 2008). The modified isoTOP-ABPP approach employed here, like other methods that rely on chemical probes to alkylate or modify cysteines in proteomes ( Klomsiri et?al., 2010; Leichert et?al.