Activity-Based Protein Profiling (ABPP) approach for target enzymes identification

From 1 L of culture at the logarithmically growth stage (OD₆₀₀~1), M. tb mc²6230 cells were harvested by centrifugation at 4,000 g for 15 min. Pellets were washed 3 times with PBS containing 0.05% Tween 80. The cell pellets were resuspended in PBS buffer at a 1:1 (w/v) ratio. The bacterial cells were then mixed with the same volume of 0.1 mm diameter glass beads (BioSpec) and disrupted during 4 min of violent shaking using Mini-Beadbeater-96 (BioSpec). The lysate was then centrifuged at 4 °C and at 12,500 g for 15 min to remove the cell debris and unbroken cells. Supernatants were adjusted to a concentration of 2 mg/mL of total proteins, snap frozen in liquid nitrogen and stored at −80 °C until further use.

M. tb mc²6230 lysates (50 µL–100 µg total proteins) were incubated with 2 μM ActivX TAMRA-FP probe (ThermoFisher Scientific) or DMSO (unlabelled control) for 90 min at room temperature and in absence of light. The reaction was stopped by adding 5X Laemmli reducing sample buffer and boiling at 95 °C for 5 min. The labelled proteins were further analyzed by SDS-PAGE electrophoresis (12% Bis-Tris gel) followed by fluorescent gel scanning (TAMRA: λ_ex 557 nm, λ_em 583 nm) and detection using the Cy^®3 filter of a ChemiDoc MP Imager (Bio-Rad). Alternatively, the gel was stained with Coomassie blue R250 staining solution and was destained with solution of 10% ethanol and 30% acetic acid.

M. tb mc²6230 lysates (500 µL–1 mg total proteins) were incubated with 2 µM ActivX Desthiobiotin-FP probe (ThermoFisher Scientific) or DMSO (unlabelled control) for 90 min at 37 °C. For inhibitor studies, lysates were pre-incubated with 580 µM CyC ₁₇ at 37 °C for 90 min prior to Desthiobiotin-FP treatment. The reaction was next stopped by adding 0.3 g of urea (10 M final concentration) to denature proteins completely. Unreacted probes were removed using Zeba Spin desalting column (7 K MWCO, ThermoFisher Scientific) and labelled proteins were further captured by 200 µg Nanolink streptavidin magnetic beads 0.8 µm (Solulink), according to the manufacturer’s instructions.

First, 20 µL of a 10 mg/mL NanoLink streptavidin magnetic beads was transferred into a 1.5 mL Eppendorf tube. The Wash Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) was then added to bring the final volume to 250 µL and the resulting mixture was mixed gently to resuspend and wash the beads. The tube was placed on a magnetic stand for 2 min. and the supernatant was discarded. The tube was removed from the magnetic stand and the beads were washed two more times with the Wash Buffer (250 µL). Each M. tb mc²6230 treated-lysate sample was enriched for labelled proteins by transfer to the previously washed beads (around 200 µg). The lysate/beads suspensions were incubated for 1 hour at room temperature with mild shaking. The tubes were then placed on the magnetic stand for 2 min to collect the beads, and the supernatant was removed. The beads containing bound, biotinylated proteins were washed three time carefully with the Wash Buffer, as described above, and resuspended in 25 µL PBS buffer pH 7.4 containing 50 mM free biotin. The resulting solution was mixed with 5X Laemmli reducing sample buffer, and heated at 95 °C for 5 min. This step allowed the recovery of the captured labelled proteins by exchanging the initially captured desthiobiotin/streptavidin complex to the greater affinity biotin/streptavidin complex.

The released proteins were resolved by SDS-PAGE at 160 V for 1 hour. The gel was stained with Coomassie blue R250 staining solution and was destained with solution of 10% ethanol; 30% acetic acid. To check for unspecific binding, a DMSO-treated lysate sample was incubated only with the streptavidin-magnetic beads in absence of Desthiobiotin-FP probe treatment, and processed as described above.