Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present Feature-Based Molecular Networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. The FBMN method brings quantitative analyses, isomeric resolution, including from ion-mobility spectrometry, into molecular networks.

Global Natural Product Social Molecular Networking (GNPS) is an interactive online small molecule-focused tandem mass spectrometry (MS 2 ) data curation and analysis infrastructure. It is intended to provide as much chemical insight as possible into an untargeted MS 2 dataset and to connect this chemical insight to the user's underlying biological questions. This can be performed within one liquid chromatography (LC)-MS 2 experiment or at the repository scale. GNPS-MassIVE is a public data repository for untargeted MS 2 data with sample information (metadata) and annotated MS 2 spectra. These publicly accessible data can be annotated and updated with the GNPS infrastructure keeping a continuous record of all changes. This knowledge is disseminated across all public data; it is a living dataset. Molecular networking-one of the main analysis tools used within the GNPS platform-creates a structured data table that reflects the molecular diversity captured in tandem mass spectrometry experiments by computing the relationships of the MS 2 spectra as spectral similarity. This protocol provides step-by-step instructions for creating reproducible, high-quality molecular networks. For training purposes, the reader is led through a 90-to 120-min procedure that starts by recalling an example public dataset and its sample information and proceeds to creating and interpreting a molecular network. Each data analysis job can be shared or cloned to disseminate the knowledge gained, thus propagating information that can lead to the discovery of molecules, metabolic pathways, and ecosystem/community interactions.

1Molecular networking has become a key method used to visualize and annotate the chemical space in 2 non-targeted mass spectrometry-based experiments. However, distinguishing isomeric compounds and

No abstract