Comprehensive LC-MS Metabolomics Workflow

The procedures for LC–MS, including analysis, peak detection, alignment, and annotation, were described elsewhere [29 (link)]. Briefly, leaf samples were prepared using methanol and MonoSpin M18 columns (GL Sciences, Tokyo, Japan). Samples were analyzed using a SHIMADZU Nexera X2 high-performance liquid chromatography (HPLC) instrument (Kyoto, Japan) with an InertSustain AQ-C18 column (2.1 × 150 mm, 3 μm particle size) (GL Sciences) connected to a Thermo Fisher Scientific Q Exactive Plus high-resolution mass analyzer (Waltham, MA, USA).
The LC–MS data obtained above were converted to mzXML format using ProteoWizard (Palo Alto, CA, USA). Peak detection, determination of ionizing states, and peak alignments were performed automatically using the data analysis software PowerGetBatch developed by the Kazusa DNA Research Institute [42 (link),43 ]. The exact mass values of the nonionized compounds calculated from the adducts were used to search candidate compounds against the UC2 chemical mass databases [44 (link)] (i.e., a combination of two databases, KNApSAcK [45 (link)] and the Human Metabolome Database [46 (link),47 (link)]) with the search program MFSearcher [48 (link)]. The LC–MS results were compiled in the Microsoft Excel file “LCMS_Result Field Data KDRI” (Supplementary File S1).