High-Resolution MS2 Metabolomics Analysis

The protocol for high-resolution Full MS data dependent MS² analyses was adapted from previous reports (Stien et al., 2019 (link), 2020 (link)). Briefly, the UHPLC system was an Ultimate 3000 (Thermo Fisher Scientific) coupled to a Q-Exactive Focus Orbitrap detector and equipped with a Phenomenex Luna Omega polar C-18 column 150 mm × 2.1 mm, 1.6 μm. Here, crude bacterial and culture medium (starch casein broth) extracts were dissolved in MeOH at a concentration of 1.5 mg/ml. Pure methanol injections were used as blanks for metabolomics. In HPLC, the solvent system was a mixture of water (solution A) with increasing proportions of acetonitrile (solution B), both solvents modified with 0.1% formic acid. Here, the gradient was as follows: 5% of solution B for 5 min before injection, then from 1 to 12 min, a linear increase of B up to 100%, followed by 100% of solution B for 8 min.