To determine protein levels in two or more different biological states (e.g. in the absence and presence of H2O2), we modified the SILAC procedure (Figure

1) in which the introduction of a stable isotope 15N into the protein mixture provides a means to quantitatively analyze two sets of protein mixtures simultaneously [31, 32]. Stable isotope-based selleck chemicals quantification relies on the premise that the relative signal intensity of two analytes that are chemically identical but different in stable isotope compositions can be resolved in a mass spectrometer, thus giving a true measure of the relative abundance of the analytes [31, 32, 34, 35]. To determine the efficiency of the labeling and incorporation of the heavy isotope, selleck SE2472 was grown in 15N-containing LB broth-like media. SE2472 appeared to grow in the normal (14N) and 15N-containing LB broth-like media as well as in the LB broth as they reach similar titers in these media (data not shown). Bacteria were harvested at different time points and https://www.selleckchem.com/products/epz015666.html the extent of 15N-labeling of Salmonella proteins was examined by MS analysis in comparison to the control 14N labeled bacteria. Growth in 15N-labeled media for 6 hours or more was sufficient to label the entire Salmonella proteome with 15N (data not shown). The proteins examined and all the peptides of each protein appeared to have

identical incorporation rate. Accordingly, all labeling experiments were carried out for at least 6 hours in this study. Figure 1 Schematic representation

of metabolic labeling of Salmonella with the 15 N isotope. Wild type-like growth phenotypes of labeled bacteria One of our main objectives in the study was to use the expression of the labeled proteins to monitor Salmonella protein levels when Salmonella is exposed to oxidative stress. Thus, it is necessary to determine whether 15N-labeled Salmonella retain the growth and oxidative Carnitine palmitoyltransferase II stress-resistant properties of the unlabeled SE2472 in vitro. 15N-labeled Salmonella appeared to grow as well as the unlabeled bacteria in LB broth (Figure 2A). No detectable difference in the colony size and morphology was observed between these two cultures. Furthermore, no difference was detected between the survival of the N14- and N15-labeled bacteria in either the LB broth-like labeling media or the LB broth in the presence of 5 mM H2O2, a concentration well below the minimal inhibition concentration (MIC) of SE2472 (20 mM) but substantially above the natural extracellular environment (Figure 2B). Figure 2 Growth analysis of S. Enteritidis SE2472. (A) Growth of normal (14N) and 15N-labeled S. Enteritidis SE2472 in LB broth. (B) The survival of normal (14N) and 15N-labeled Salmonella grown in LB broth-like labeling media after exposure to H2O2, compared to the survival of the same cultures grown in LB broth after exposure to H2O2 (inset).