Figure 3.

Activity based protein profiling (ABPP) workflow for the identification of the proteins covalently bound to CyC₁₇ inhibitor. Cell lysates of M. tb mc² 6230 were either (A) pre-treated with CyC₁₇ prior to incubation with Desthiobiotin-FP probe or (B) incubated with Desthiobiotin-FP alone. Both samples were further treated with streptavidin-magnetic beads for the capture and enrichment of labelled proteins. (C) Uncompetitive binding assay using streptavidin-magnetic beads on cell lysate. (D) Detection of all potential serine/cysteine enzymes in total cell lysate using fluorescent TAMRA-FP probe. (E) Equal amounts of proteins obtained in A to D were separated by SDS-PAGE and visualized by Coomassie staining (right panel – lanes A–C) or in-gel fluorescence (left panel - lane D: TAMRA detection). Enzymes whose labelling is impeded because of the presence of CyC₁₇ in the active-site are circled in red and shown by arrowheads. The corresponding bands were excised form the gel and subjected to triptic digestion and tandem mass spectrometry analysis. The SDS gel presented in panel E is representative of three independent ABPP experiments.