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Oxidative dysfunction in the metabolism has long been implicated in diverse biological disorders. Although a substantial number of metabolic enzymes are targeted for inactivation by oxidative stress, identifying those targets remains difficult due to a lack of comprehensive observations of the metabolism acting through the stress response. We herein developed a metabolomics strategy using integrative liquid chromatography‐mass spectrometry (LC‐MS) and observing rapid metabolomic changes in response to hydrogen peroxide (H₂O₂)‐induced oxidative stress in HeLa cells. Among the many metabolite changes detected, the most characteristic metabolites uniquely indicated carnitine palmitoyltransferase‐1 (CPT1), the critical enzyme for mitochondrial β‐oxidation of long‐chain fatty acids, to be a target for oxidative inactivation. We showed that the enzymatic activity of CPT1 significantly declined by H₂O₂ in …