Samples were extracted with 80% methanol and analyzed on an Agilent 1200 Rapid Resolution Liquid Chromatography (RRLC) system equipped with a ZORBAX SB-Aq column (2.1×100 mm, 1.8 µm) couple with an Agilent 6510 Q-TOF performed in positive ionization mode. The column temperature was set at 50 °C and the flow rate was 0.3 mL/min. Water with 0.1% formic acid and acetonitrile were used as mobile phases A and B, respectively. Mass spectrometric analysis was performed with nitrogen was used as the nebulizer gas at 45 psi and as drying gas at 350 °C with a flow rate of 9 l/min. The ESI spray voltage was 4,000 V and the voltage of the fragmentor was 175 V. Raw data files were exported in netCDF format and were processed as described by Kim and colleagues (2011) (link), including peak detection, retention time correction, and annotation of isotope and adduct ions using the bioconductor XCMS and CAMERA packages (http://www.bioconductor.org/). The peak areas were normalized to the total peak area, and peaks that were present in less than 80% samples were discarded from the total peak list to minimize the number of missing values. Peaks with CV values > 20% in quality control (QC) samples were deleted to ensure the reliability of the data. The known features were then used for the following analysis, and p < 0.05 was the standard for differential regulation.