Feature annotation was performed by using our spectral database first according to accurate measured masses and chromatographic retention times [61,67,68] and then according to publicly available databases KEGG, PAMDB, HMDB, and METLIN [34,69,70,71] solely using accurate masses. This data-based analysis allowed putative annotation of 271 metabolites (Supplementary Table S2). Metabolite identification was further confirmed for discriminant metabolites LC-MS/MS experiments using a Dionex Ultimate chromatographic system combined with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) under non-resonant collision-induced dissociation conditions using higher-energy C-trap dissociation (HCD). To be identified, metabolites had to match at least two orthogonal criteria (among accurate measured mass, retention time, and MS/MS spectrum) to those of an authentic chemical standard analyzed under the same analytical conditions, as proposed by the Metabolomics Standards Initiative [72]. In the absence of an available authentic chemical standard, metabolites of interest were only considered as putatively annotated based on accurately measured masses and interpretation of the MS/MS spectra when available as described by Aros-Calt et al. 2015 [61]. Under these conditions, up to 51 discriminant metabolites were characterized: 30 had accurate masses, retention times and MS/MS matching those of an authentic standard, 3 were putatively annotated by matching their MS/MS spectra to those from the METLIN public database or showed MS/MS spectra consistent with both the proposed structures and the spectra of structural homologues, 1 shared accurate mass and retention time with an authentic standard, and 17 compounds were only annotated based on their accurate masses (Supplementary Table S3).

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