Untargeted Metabolomics Data Processing UsingFragHub andMS-DIAL Softwares v1

This protocol presents a comprehensive approach for untargeted metabolomics data analysis using the open-source software platforms FragHub and Mass Spectrometry–Data Independent AnaLysis software (MS-DIAL). The workflow is designed for LC-MS/MS-based metabolomics, focusing on optimizing the detection, identification, and annotation of metabolites. FragHub integrates multiple mass spectral libraries and formats, including .MSP, .MGF, .JSON, and .CSV, using RDKit to harmonize metadata, which improves the accuracy and reliability of metabolite annotation. It enables users to unify various open mass spectral libraries (OMSLs), facilitating enhanced metabolite identification. MS-DIAL, a versatile tool supporting various MS vendors and instruments, enables peak detection, deconvolution, and spectral matching against libraries. MS-DIAL allows efficient precursor ion detection, chromatogram deconvolution, and compound identification through retention time, accurate mass, and MS/MS spectral matching. Together, FragHub and MS-DIAL provide a robust framework for untargeted metabolomics data processing, enhancing the accuracy and reproducibility of metabolite discovery.