Comprehensive Metabolomics Profiling by LC-MS

Metabolomics profiling was performed by Ultimate 3000 LC combined with Q Exactive MS (Thermo Fisher Scientific, CA, United States) as reported in our recent publication (Aleidi et al., 2021 (link)). Briefly, extracted metabolites were first separated using an ACQUITY UPLC HSS T3 (100 × 2.1 mm 1.8 μm) and a mobile phase composed of solvent A (0.05% formic acid-water) and solvent B (ACN) with a gradient elution over 16 min applied at 300 μl/min flow rate. MS spectra were acquired in full MS scan in the range m/z 50–1,500, with 25,000 enhanced mass resolution and a frequency 15 spectra per second. The capillary voltage was 3000 and 3200 V for positive and negative ionization modes, respectively. The fragmentation was achieved for MSMS experiments at 175 V, with nebulizer gas at 35 bsi, and gas temperature 450°C. Chromatographic and MS parameters (under positive and negative ionization modes) were kept as reported previously (Aleidi et al., 2021 (link)). Pooled samples prepared the quality control (QC) sample. A QC injection was performed every 10 LC-MS sample runs. In total, there were 18 QC samples injected and analyzed.