MolFind²: A Protocol for Acquiring and Integrating MS³ Data to Improve In Silico Chemical Structure Elucidation for...

Structure elucidation of metabolites (<1000 Da) in biofluids is extremely challenging due to the diversity and complexity of chemical structure space. Generally, due to lack of reference tandem mass data (MS²), in silico fragmenters are used to rank candidates acquired from chemical databases as a function on how well they explain an experimental collision-induced dissociation spectrum. However, multistage fragmentation data (i.e., MS³) have not been adequately utilized in current metabolomics structure elucidation pipelines. To address this shortcoming, here we describe an experimental (nontargeted direct infusion ion mobility-mass spectrometry-based) and computational workflow to acquire and utilize multistage mass (MS³) spectrometry data for database-assisted structure elucidation.