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A primary goal of metabolomics is to identify all biologically important metabolites. One powerful approach is liquid chromatography-high resolution mass spectrometry (LC-MS), yet most LC-MS peaks remain unidentified. Here, we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. We consider all experimentally observed ion peaks together, and assign annotations to all of them simultaneously so as to maximize a score that considers properties of peaks (known masses, retention times, MS/MS fragmentation patterns) as well network constraints that arise based on mass difference between peaks. Global optimization results in accurate peak assignment and trackable peak-peak relationships. Applying this approach to yeast and mouse data, we identify a half-dozen novel metabolites, including thiamine and taurine derivatives. Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to annotate untargeted metabolomics data, revealing novel metabolites.

Untargeted metabolomics can detect more than 10,000 peaks in a single LC-MS run. The correspondence between these peaks and metabolites, however, remains unclear. Here we introduce a Peak Annotation and Verification Engine (PAVE) for annotating untargeted microbial metabolomics data. The workflow involves growing cells in 13 C and 15 N isotope-labeled media to identify peaks from biological compounds and their carbon and nitrogen atom counts. Improved de-isotoping and de-adducting is enabled by algorithms that integrate positive mode, negative mode and labeling data. To distinguish metabolites and their fragments, PAVE experimentally measures the response of each peak to weak in-source collision induced dissociation, which increases the peak intensity for fragments while decreasing it for their parent ions. The molecular formulae of the putative metabolites are then assigned based on database searching using both m/z and C/N atom counts. Application of this procedure to S. cerevisiae and E. coli revealed that more than 80% peaks do not label, i.e. are environmental contaminants. More than 70% of the biological peaks are isotopic variants, adducts, fragments, or mass spectrometry artifacts yielding ~2,000 apparent metabolites across the two organisms. About 650 match to a known metabolite formula based on m/z and C/N atom counts, with 220 assigned structures based on MS/MS and/or retention time to match to authenticated standards. Thus, PAVE enables systematic annotation of LC-MS metabolomics data, with only ~ 4% of peaks annotated as apparent metabolites.

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