GNPS - Charting Unknown Metabolic Reactions by Mass Spectrometry-Resolved Stable-isotope Tracing Metabolomics

Metabolic reactions play important roles in live organisms such as providing energy, transmitting signals, and synthesizing biomacromolecules. Charting unknown metabolic reactions in cells is hindered by limited technologies, restricting the holistic understanding of cellular metabolism. Using the mass spectrometry-resolved stable-isotope tracing metabolomics, we developed an isotopologue similarity networking strategy, namely IsoNet, to effectively deduce new metabolic reactions. The strategy uncovered ~300 new metabolic reactions in living cells and mice. Specifically, we elaborately charted the metabolic reaction network related to glutathione, unveiling three previously unreported reactions nestled within glutathione metabolism. Among these, a transsulfuration reaction, synthesizing gamma-glutamyl-seryl-glycine directly from glutathione, underscored the role of glutathione as a sulfur donor. Functional metabolomics studies systematically characterized biochemical effects of new reactions in glutathione metabolism, showcasing their diverse functions in regulating cellular metabolism. Overall, these newly uncovered metabolic reactions fill gaps in the metabolic network maps, facilitating exploration of uncharted territories in cellular biochemistry.