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

Abstract

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.