A protocol for high‐throughput, untargeted forest community metabolomics using mass spectrometry molecular networks

PREMISE OF THE STUDY: We describe a field collection, sample processing, and ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) instrumental and bioinformatics method developed for untargeted metabolomics of plant tissue and suitable for molecular networking applications. METHODS AND RESULTS: A total of 613 leaf samples from 204 tree species was collected in the field and analyzed using UHPLC‐MS/MS. Matching of molecular fragmentation spectra generated over 125,000 consensus spectra representing unique molecular structures, 26,410 of which were linked to at least one structurally similar compound. CONCLUSIONS: Our workflow is able to generate molecular networks of hundreds of thousands of compounds representing broad classes of plant secondary chemistry and a wide range of molecular masses, from 100 to 2500 daltons, making possible large‐scale comparative metabolomics, as well as studies of chemical community ecology and macroevolution in plants.