C18 silica particles

The LC–MS/MS measurements were conducted with a Q-Exactive tandem mass spectrometer and an Easy-nLC 1000 nano HPLC system (Thermo Fisher Scientific). The trap column used for the nano HPLC was a 2 cm × 75 μm capillary column packed with 3 μm C18-silica particles (Thermo Fisher Scientific), and the separation column was a 12.5 cm × 75 μm capillary column packed with 3 μm C18-silica particles (Nikkyo Technos, Japan). The flow rate for the nano HPLC was 300 nL/min. The separation was conducted using a 10−40% linear acetonitrile gradient over 70 min, in the presence of 0.1% formic acid. The nanoLC–MS/MS data were acquired in the data-dependent acquisition (DDA) mode controlled by the Xcalibur 4.0 program (Thermo Fisher Scientific). The DDA settings were as follows: the resolution was 70 000 for a full MS scan and 17 500 for an MS2 scan; the AGC target was 3.0E6 for a full MS scan and 5.0E5 for an MS2 scan; the maximum IT was 60 ms for both the full MS and MS2 scans; the full MS scan range was 310–1500 m/z; and the top 10 signals in each full MS scan were selected for the MS2 scan. The DDA measurement was performed three times for each sample, and two biological replicates were measured in each condition except for the set of antibiotics experiment.