Multi-kingdom Metabolite Extraction Protocol

Human urine from volunteers was prepared using an established urine metabolomics protocol [18 (link)]. Urine was aliquoted into 200 µL samples and prepared, including the depletion of urea with urease, precipitation of urease with methanol and extraction of metabolites. The plant metabolome extract was generated from seagrass leaves (Posidonia oceanica, collected in the Mediterranean sea, the island of Elba) and was prepared using the method for global plant metabolomics [6 (link)]. Briefly, shock-frozen leaf tissue was ground with a mortar and pestle (all cooled with liquid N₂), 100 mg powder was extracted with methanol and strong agitation, chloroform and water were added and the sample mixed. After phase-separation, the upper layer was taken for metabolite analysis. Lysogeny broth (Sigma Aldrich) was prepared after the manufacturer’s recipe, excluding the addition of salts, and 100 µL was used as a sample for the yeast cell metabolite extract. Animal tissue (common earthworm, Lumbricus terrestris) was collected and extracted as described previously [19 (link)]. Briefly, the shock-frozen whole animal was ground with a mortar and pestle (all liquid N₂ cooled), 50 mg powder was extracted as described above for the plant tissue. All final extracts were dried under vacuum (Extractor Plus^®, Agilent; settings: 30 °C, 2 h, 1000 rpm, VAQ).