MZmine 2 data preprocessing parameters

Networks were generated using the methodology previously described by Olivon et al.²³. Mass detection was realized keeping the noise level at 3.0E5 for MS level 1 and 0 for MS level 2. ADAP chromatogram builder was employed using a minimum group size of scans of 5, a group intensity threshold of 3.0 × 10⁵, and an m/z tolerance of 0.01. ADAP wavelet deconvolution algorithm was used with the following standard settings: S/N threshold of 10, minimum feature height of 3.0 × 10⁵, coefficient/area threshold of 10, peak duration range of 0.05–0.6 min, and RT wavelet range of 0.01–0.15. MS2 scans were paired using a m/z tolerance range of 0.01 Da and a RT tolerance range of 0.1 min. Isotopologues were grouped using the isotopic peak grouper algorithm [m/z tolerance of 20 ppm, RT tolerance of 0.1 min, maximum charge of 3]. For peak alignment, step was performed using Join aligner module [m/z tolerance of 20 ppm, weight for m/z of 75, RT tolerance of 0.1 min, weight for RT of 25]. The resulting peak list was filtered to exclude the first 70 s (to remove molecules not retained in the column) and then gap-filled with the “same RT and m/z range gap filler” module [m/z tolerance of 20 ppm].

Eventually, .mgf preclustered spectral data files and their corresponding .csv metadata files (for RT, areas, and formula integration) were exported using the dedicated “Export for GNPS” and “Export to CSV file” built-in options.