The detection of induced proteins after introduction of specific substrates in culture is of high interest for a comparative description of organisms growing under different conditions. In this study, protein-based stable isotope probing (Protein-SIP) is used for a fast and reliable detection of newly synthesized proteins in a substrate shift experiment. Therefore, Pseudomonas putida ML2 cells precultured on (12)C-acetate and (12)C-benzene, respectively, were incubated with (13)C-benzene as a stable-isotope-labeled substrate. Protein samples from early to stationary growth phase were separated by one-dimensional gel electrophoresis (1-DE), subsequently tryptically digested, and analyzed by UPLC Orbitrap MS/MS measurements. Identified peptides from proteins involved in aerobic benzene degradation as well as from housekeeping proteins were chosen to calculate the labeling ratio (proportion of labeled protein to total protein) at different time points. A comparison of parameters from a nonlinear regression analysis of the calculated data enabled a clear differentiation between induced and constitutively expressed proteins. Thus, Protein-SIP has proven to be a valuable tool for quantitative analysis of induced proteins in substrate shift experiments.
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