Detection of\nInverse Stable Isotopic Labeling in Untargeted\nMetabolomic Data

Stable isotopic labeling is a powerful\ntool for determining\nthe\nbiosynthetic origin of metabolites and for discovering natural products\nthat incorporate precursors of interest. When isotopically substituted\nprecursors are not available commercially or synthetically, inverse\nstable isotopic labeling (InverSIL) is a useful alternative. With\nInverSIL, an organism is grown on an isotopically substituted medium\nand then fed precursors of natural isotopic abundance which can be\ntracked by mass spectrometry, thereby bypassing issues with precursor\navailability. Currently, there is no automated way to identify precursor\nincorporation in untargeted metabolomic data using InverSIL without\nspecifying an expected change in the mass-to-charge ratio of metabolites\nthat have incorporated the precursor. This makes it difficult to identify\nunknown natural products that may incorporate portions of precursors\nof interest using new biochemistry or to rapidly identify incorporation\nof multiple precursors into different metabolites simultaneously.\nTo address this, we developed a new, robust workflow for the automated\nidentification of inverse labeling in untargeted metabolomic data.\nWe then use this method to identify metabolites that incorporate <i>para</i>-aminobenzoic acid and different portions of l-methionine, including in the same sample, and in the process discover\nthe likely biosynthetic origin for the C-7 and C-9 methyl groups of\nthe pterin portion of dephosphotetrahydromethanopterin, a C1 transfer\ncoenzyme used by methylotrophic bacteria. This workflow can be applied\nin the future to streamline the use of the versatile InverSIL approach\nfor natural product and metabolism research.