Proteomic Analysis of Bacterial Extracts

Bacterial pellets were diluted in 1× LDS (Thermo) buffer at a ratio of 7.5 μL/mg. They were heated for 10 min at 99°C, sonicated for 10 min, and then subjected to bead-beating lysis as previously described (50 (link)). The extracted proteins (25 μL) were subjected to denaturing electrophoresis onto a NuPAGE 4 to 12% Bis-Tris gel (Thermo) for 4 min. For each sample, a single gel band containing the whole cellular proteome was excised and processed as described previously (51 (link)) for in-gel trypsin proteolysis. Peptides were analyzed using a Q-Exactive HF tandem mass spectrometer (Thermo) coupled to an Ultimate 3000 nanoRSLC nanoLC system (Thermo) in data-dependent acquisition mode as previously described in reference 52 (link).