Molecular formula discovery via bottom-up MS/MS interrogation

Abstract A substantial fraction of metabolic features remains undetermined in mass spectrometry (MS)-based metabolomics. Here we present bottom-up tandem MS (MS/MS) interrogation to illuminate the unidentified features via accurate molecular formula annotation. Our approach prioritizes MS/MS-explainable formula candidates, implements machine-learned ranking, and offers false discovery rate estimation. Compared to the existing MS1-initiated formula annotation, our approach shrinks the formula candidate space by 42.8% on average. The superior annotation accuracy of our bottom-up interrogation was demonstrated on reference MS/MS libraries and real metabolomics datasets. Applied on 155,321 annotated recurrent unidentified spectra (ARUS), our approach confidently annotated >5,000 novel molecular formulae unarchived in chemical databases. Beyond the level of individual metabolic features, we combined bottom-up MS/MS interrogation with global peak annotation. This approach reveals peak interrelationships, allowing the systematic annotation of 37 fatty acid amide molecules in human fecal data, among other applications. All bioinformatics pipelines are available in a standalone software, BUDDY ( https://github.com/HuanLab/BUDDY/ ).