Metabolite analysis
An UltiMate 3000 U-HPLC system (Dionex) was used to create a 45 minutes
linear gradient of 5-35% (v/v) acetonitrile in 0.1% (v/v) formic acid
(FA) in water at a flow rate of 0.19 mL min-1. 5 µL of
each extract was injected and compounds were separated on a Luna C18
column (2.0 x 150 mm, 3µm; Phenomenex) maintained at 40 °C (De Voset al. 2007). The detection of compounds eluting from the column
was performed with a Q-Exactive Plus Orbitrap FTMS mass spectrometer
(Thermo Scientific). Full scan MS data were generated with electrospray
in switching positive/negative ionization mode at a mass resolution of
35,000 (FWHM at m/z 200) in a range of m/z 95-1350. Subsequent MS/MS
experiments for identification of selected metabolites were performed
with separate positive or negative electrospray ionization at a
normalized collision energy of 27 and a mass resolution of 17,500. The
ionization voltage was optimized at 3.5 kV for positive mode and 2.5 kV
for negative mode; capillary temperature was set at 250 °C; the
auxiliary gas heater temperature was set to 220 °C; sheath gas,
auxiliary gas and the sweep gas flow were optimized at 36, 10 and 1
arbitrary units, respectively. Automatic gain control was set at
3e6 and the injection time at 100 ms. External mass
calibration with formic acid clusters was performed in both positive and
negative ionization modes before each sample series.