Systems-level analysis of isotopic labeling in untargeted metabolomic data by X13CMS.

Identification of previously unreported metabolites (so-called 'unknowns') in untargeted metabolomic data has become an increasingly active area of research. Considerably less attention, however, has been dedicated to identifying unknown metabolic pathways. Yet, for each unknown metabolite structure, there is potentially a yet-to-be-discovered chemical transformation. Elucidating these biochemical connections is essential to advancing our knowledge of cellular metabolism and can be achieved by tracking an isotopically labeled precursor to an unexpected product. In addition to their role in mapping metabolic fates, isotopic labels also provide critical insight into pathway dynamics (i.e., metabolic fluxes) that cannot be obtained from conventional label-free metabolomic analyses. When labeling is compared quantitatively between conditions, for example, isotopic tracers can enable relative pathway activities to be inferred. To discover unexpected chemical transformations or unanticipated differences in metabolic pathway activities, we have developed X13CMS, a platform for analyzing liquid chromatography/mass spectrometry (LC/MS) data at the systems level. After providing cells, animals, or patients with an isotopically enriched metabolite (e.g., 13C, 15N, or 2H), X13CMS identifies compounds that have incorporated the isotopic tracer and reports the extent of labeling for each. The analysis can be performed with a single condition, or isotopic fates can be compared between multiple conditions. The choice of which metabolite to enrich and which isotopic label to use is highly context dependent, but 13C-glucose and 13C-glutamine are often applied because they feed a large number of metabolic pathways. X13CMS is freely available.