Protocol for community‐created public MS/MS reference spectra within the Global Natural Products Social Molecular Networking infrastructure

Rationale A major hurdle in identifying chemicals in mass spectrometry experiments is the availability of tandem mass spectrometry (MS/MS) reference spectra in public databases. Currently, scientists purchase databases or use public databases such as Global Natural Products Social Molecular Networking (GNPS). The MSMS‐Chooser workflow is an open‐source protocol for the creation of MS/MS reference spectra directly in the GNPS infrastructure. Methods An MSMS‐Chooser Sample Template is provided and completed manually. The MSMS‐Chooser Submission File and Sequence Table for data acquisition were programmatically generated. Standards from the Mass Spectrometry Metabolite Library (MSMLS) suspended in a methanol–water (1:1) solution were analyzed. Flow injection on an LC/MS/MS system was used to generate negative and positive mode data using data‐dependent acquisition. The MS/MS spectra and Submission File were uploaded to MSMS‐Chooser workflow in GNPS for automatic selection of MS/MS spectra. Results Data acquisition and processing required ~2 h and ~2 min, respectively, per 96‐well plate using MSMS‐Chooser. Analysis of the MSMLS, over 600 small molecules, using MSMS‐Chooser added 889 spectra (including multiple adducts) to the public library in GNPS. Manual validation of one plate indicated accurate selection of MS/MS scans (true positive rate of 0.96 and a true negative rate of 0.99). The MSMS‐Chooser output includes a table formatted for inclusion in the GNPS library as well as the ability to directly launch searches via MASST. Conclusions MSMS‐Chooser enables rapid data acquisition, data analysis (selection of MS/MS spectra), and a formatted table for inspection and upload to GNPS. Open file‐format data (.mzML or.mzXML) from most mass spectrometry platforms containing MS/MS spectra can be processed using MSMS‐Chooser. MSMS‐Chooser democratizes the creation of MS/MS reference spectra in GNPS which will improve annotation and strengthen the tools which use the annotation information.