A guide to reverse metabolomics-a framework for big data discovery strategy

Untargeted metabolomics is evolving into a field of big data science. There is a growing interest within the metabolomics community in mining tandem mass spectrometry (MS/MS)-based data from public repositories. In traditional untargeted metabolomics, samples to address a predefined question are collected and liquid chromatography with MS/MS data are generated. We then identify metabolites associated with a phenotype (for example, disease versus healthy) and elucidate or validate their structural details (for example, molecular formula, structural classification, substructure or complete structural annotation or identification). In reverse metabolomics, we start with MS/MS spectra for known or unknown molecules. These spectra are used as search terms to search public data repositories to discover phenotype-relevant information such as organ/biofluid distribution, disease condition, intervention status (for example, pre- and postintervention), organisms (for example, mammals versus others), geography and any other biologically relevant associations. Here we guide the reader through a four-part process: (1) obtaining the MS/MS spectra of interest (Universal Spectrum Identifier) and (2) Mass Spectrometry Search Tool searches to find the files associated with the MS/MS that are in available databases, (3) using the Reanalysis Data User Interface framework to link the files with their metadata and (4) validating the observations. Parts 1-3 could take from hours to days depending on the method used for collecting MS/MS spectra. For example, we use MS/MS spectra from three small molecules: phenylalanine-cholic acid (a microbially conjugated bile acid), phenylalanine-C4:0 and histidine-C4:0 (two N-acyl amides). We leverage the Global Natural Products Social Molecular Networking-based framework to explore the microbial producers of these molecules and their associations with health conditions and organ distributions in humans and rodents.