Microtofcontrol

LC-(Q) TOF/MS analysis was performed on an UltiMate 3000 system (Dionex, Sunnyvale, CA, USA) comprising a pump, a UV detector (208 nm), and an autosampler cooled to 4 °C with an Inertsil ODS-3v column [5 µm, 4.6 × 250 mm (GL Science Inc., Tokyo, Japan)]. The two samples (each 50 µL) were directly injected into the HPLC column. The separation of tomatines was eluted with 20 mM ammonium acetate/acetonitrile (65:35, v/v) at a flow rate of 700 μL/min at 30 °C, and a MicroQ-TOF III mass spectrometer with an electrospray interface (ESI) source (Bruker Daltonics, Bremen, Germany). The interface voltage and current were 4.50 kV and 1.6 μA for the negative-ion mode. The flow rate of nebulizing gas was 1.5 L/min, and the N₂ drying pressure was 0.2 M Pa. The curved desorption line and heat block temperature were both at 200 °C. The detector voltage of the TOF analyzer was 1.68 kV. Ultrahigh-purity argon was used as the collision gas for collision-induced dissociation experiments. The relative energy in collisions was 100%. The sample injection volume was 50 μL. A direct valve was set to transmit and divert the HPLC eluent to waste. Mass spectral data were collected from m/z 160–1100. Data acquisition and processing were carried out with micrOTOFcontrol and dataAnalysis 4.0 (Bruker Daltonics, Bremen, Germany) software.

Metabolite identification was done by MetabolitePilot™ software version 2.0 (SCIEX). The MS/MS spectrum of IVM was exported as text files by the PeakView software (SCIEX) and imported to MetabolitePilot™ software (SCIEX) as a reference spectrum for creating the IVM library. Raw data files (.wiff) of metabolite sample analyses were imported to MetabolitePilot™ software and compared against the IVM‐library peak finding strategies as described in the supplementary material (Supplement Appendix S1). For the LC‐SPE‐NMR/MS system, the HPLC was operated by Hystar 3.2 (Bruker Daltonics), mass spectrum acquired by Microtof control (Bruker Daltonics), and the NMR spectrometer was operated by Topspin 3.5 (Bruker Biospin). The Complete Molecular Confidence – Structure Elucidation (CMC‐se) software version 2.6.1 (Bruker Biospin) was used for structure elucidation.

All of the mass spectrometry data were collected using Bruker Daltonics micrOTOF control and processed and analysed using the Data Analysis Software. The Uniprot database was downloaded and integrated into the Mascot search engine, version 2.3.01, through its database maintenance unit. The parameters were set as follows: trypsin was specified as the digestion enzyme, cysteine carbamidomethylation as a fixed modification, iTRAQ 8Plex on the N-terminal residue, iTRAQ 8Plex on tyrosine (Y), iTRAQ 8Plex on lysine (K), glutamine as pyroglutamic acid, and oxidation on methionine (M) as a variable modification. The tolerance settings for peptide identification in the Mascot searches were 0.05 Da for MS and 0.05 Da for MS/MS. The Mascot search results were exported into a DAT FILE and normalized and quantified using the Scaffold version 3.0 Software. Protein quantification was carried out based on a unique peptide. A cut-off of a 1.5-fold change was chosen, and proteins with quantification ratios of 1.5 for low (<0.67) or high (>1.5) relative protein levels were considered as differentially regulated.