Bacillus subtilis metabolomics

Bacillus subtilis metabolomics:

Analyses were performed using an UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a QTRAP MS (AB 6500+, ABSciex) in Shanghai Applied Protein Technology Co., Ltd. The analytes were separated on HILIC (Waters UPLC BEH Amidecolumn, 2.1 mm × 100 mm, 1.7μm) and C18 columns (Waters UPLC BEH C18-2.1x100 mm, 1.7 μm).

For HILIC separation, the column temperature was set at 35 ℃; and the injection volume was 2 μL. Mobile phase A: 90%H2O + 2 mM ammonium formate + 10% acetonitrile , mobile phase B: 0.4% formic acid in acetonitrile. A gradient (85% B at 0-1 min, 80% B at 3-4 min, 70% B at 6 min, 50% B at 10-15.5 min, 85% B at 15.6 -23 min ) was then initiated at a flow rate of 300μL/min.

For RPLC separation, the column temperature was set at 40℃, and the injection volume was 2 μL. Mobile phase A: 5 mMammonium acetate in water, mobile phase B: 99.5% acetonitrile.A gradient (5% B at 0 min, 60% B at 5 min, 100% B at 11-13min, 5% B at 13.1-16 min ) was then initiated at a flow rate of 400 μL/min. The sample was placed at 4 ℃ during the wholeanalysis process.

6500+ QTRAP (AB SCIEX) was performed in positive and negative switch mode. The ESI positive source conditions were asfollows: Source temperature: 580℃; Ion Source Gas1 (GS1): 45; Ion Source Gas2 (GS2): 60; Curtain Gas (CUR): 35; IonSprayVoltage(IS): +4500 V; The ESI negative source conditions were as follows: Source temperature: 580℃; Ion Source Gas1(GS1): 45; Ion Source Gas2 (GS2): 60; Curtain gas (CUR): 35; IonSpray Voltage(IS): -4500 V. MRM method was used for massspectrometry quantitative data acquisition. The MRM ion pairs are showed in the attached file. A polled quality control (QC)samples were set in the sample queue to evaluate the stability and repeatability of the system.