Positive or negative ion mode calibration mixes

Twenty uL of each extracted supernatant sample were injected into an ultra-high-performance liquid chromatography (UHPLC) system (Ultimate 3000, Thermo): samples were loaded onto a Reprosil C18 column (2.0mm× 150 mm, 2.5 μm-DrMaisch, Germany) for metabolite separation. Chromatographic separations were made at flow rate of 0.2 ml/min. A 0–100% linear gradient of solvent A (ddH2O, 0.1% formic acid) to B (acetonitrile, 0.1% formic acid) was employed over 20 min, returning to 100% A in 2 min and holding solvent A for a 6-min post time hold. The UHPLC system was coupled online with a Q Exactive mass spectrometer (Thermo Fisher, Rockford, IL) scanning in full MS mode (2 μ scans) at resolution of 70,000 in the 67 to 1,000 m/z range, with 3.8 kV spray voltage, 40 sheath gas, and 25 auxiliary gas. The system was operated in positive ion mode. Calibration was performed before each analysis against positive or negative ion mode calibration mixes (Thermo Fisher) to ensure error of the intact mass within the sub ppm range. Metabolite assignments were performed using MAVEN v5.2 [40 (link)]. Each replicate was analysed separately and a p-value < 0.01 was used to infer significance for all abundance comparisons between sets of triplicates.

Twenty μL of extracted supernatant samples was injected into an ultra-high-performance liquid chromatography (UHPLC) system (Ultimate 3000, Thermo) and run on a positive mode: samples were loaded on to a Reprosil C18 column (2.0 mm× 150 mm, 2.5 μm—Dr Maisch, Germany) for metabolite separation. Chromatographic separations were made at a column temperature of 30 °C and a flow rate of 0.2 mL/min. For positive ion mode (+) MS analyses, a 0–100% linear gradient of solvent A (ddH2O, 0.1% formic acid) to B (Acetonitrile, 0.1%formic acid) was employed over 20 min, returning to 100% A in 2 min and holding solvent A for a 1-min post time hold. Acetonitrile, formic acid, and HPLC-grade water and standards (≥98% chemical purity) were purchased from Sigma Aldrich. The UHPLC system was coupled online with a Q Exactive mass spectrometer (Thermo) scanning in full MS mode (2 μscans) at resolution of 70,000 in the 67 to 1000 m/z range, a target of 1106 ions and a maximum ion injection time (IT) of 35ms with 3.8 kV spray voltage, 40 sheath gas, and 25 auxiliary gas. The system was operated in positive ion mode. Calibration was performed before each analysis against positive or negative ion mode calibration mixes (Pierce, Thermo Fisher, Rockford, IL) to ensure error of the intact mass within the sub ppm range.