Tomato Case Study for Validation of DeepMASS

The supernatants were pipetted and dried in a LABCONCO CentriVap vacuum centrifugal concentrator (We Brothers Scientific (Pvt.) Ltd., MO, USA) and resuspended in 100 µL of reconstitution buffer (MeOH:H2O = 80:20, v/v). A 10 µL aliquot of the supernatant from each sample was pipetted and mixed as QC samples.Then, extracts were analyzed by a U3000 UHPLC system coupled with a Q Exactive orbitrap mass spectrometer (Thermo Fisher Scientific Ltd., CA, USA) equipped with a HESI probe under both positive and negative modes.

Extracts were separated by an ACQUITY UPLC™ BEH C18 column (Waters Ltd., MA, USA) (150 × 2.1 mm, 2.6 µm). The injection volume was set at 1 µL, and the flow rate was set at 300 μL/min. Solvents for the mobile phase were 0.05% formic acid in acetonitrile (A) and 0.1% formic acid in water (B).

The elution gradient was as follows: 0-25 min, linear gradient 40%-100% A, followed by column washing with 100% B and 100% A. Data with mass ranges of m/z 80-1200 and m/z 80-1200 were acquired in both positive ion mode and negative ion mode separately, with data-dependent MS/MS acquisition. The full scan and fragment spectra were collected with resolutions of 70,000 and 17,500, respectively. Data preprocessing and MS/MS extraction were conducted through MS-DIAL(Tsugawa et al., 2015).

The data processing parameter settings were as follows: peak height of 20000, mass slice of 0.1 Da, alignment retention time tolerance of 0.05 min. Subsequently, the MS/MS files were exported and inputted into the DeepMASS v2 software.