An Untargeted Approach for Revealing Electrophilic Metabolites.

Reactive electrophilic intermediates such as coenzyme A esters play central roles in metabolism but are difficult to detect with conventional strategies. Here, we introduce hydroxylamine-based stable isotope labeling to convert reactive electrophilic intermediates into stable derivatives that are easily detectable LC-MS. In the model system , parallel treatment with NHOH and NHOH revealed >1000 labeled metabolites, e.g., derived from peptide, fatty acid, and ascaroside pheromone biosyntheses. Results from NHOH treatment of a pheromone biosynthesis mutant, , suggested upregulation of thioesterase activity, which was confirmed by gene expression analysis. The upregulated thioesterase contributes to the biosynthesis of a specific subset of ascarosides, determining the balance of dispersal and attractive signals. These results demonstrate the utility of NHOH labeling for investigating complex biosynthetic networks. Initial results with and human cell lines indicate applicability toward uncovering reactive metabolomes in diverse living systems.