Metabolite Profiling of Culture Supernatants

Analytical samples for metabolite profiling were prepared by diluting culture supernatant broth extracts (0.2 ml) with HPLC-grade water (0.2 ml). The UHPLC-LTQ-Orbitrap-MS/MS system used was equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, USA) coupled with an autosampler and a column compartment. Chromatographic separation was performed on a Phenomenex KINETEX C18 column (100 mm × 2.1 mm, 1.7μm; Torrance, USA) with an injection volume of 5 μl. The column temperature was set to 40°C, and the flow rate was 0.3 ml/min. The mobile phases consisted of 0.1% v/v formic acid in water (A) and 0.1% v/v formic acid in acetonitrile (B). The gradient parameters were set as described by Kwon et al. [19 (link)]. The MS data were collected in the range of 100–1500 m/z (under negative- and positive-ion mode) using an Orbitrap Velos ProTM system, which is combined with an Ion-Trap Mass Spectrometer (Thermo Fisher Scientific) coupled with a HESI-II probe. The probe heater and capillary temperatures were set to 300°C and 350°C, respectively. The capillary voltage was set to 2.5 kV in negative mode (positive mode, 3.7 kV).