Resolving the subcellular redox landscape of Coenzyme Q

Coenzyme Q (CoQ) is an essential electron carrier and lipophilic antioxidant whose biological functions depend on its redox state. However, accurate measurement of reduced and oxidized CoQ is hindered by rapid ex vivo oxidation during sample preparation and analysis. Here, we develop a dual-isotope-based oxidation-correction LC-MS/MS platform that enables accurate quantification of CoQ redox status across diverse biological matrices. Coupled with rapid subcellular fractionation, this approach resolves cytosolic and mitochondrial CoQ pools and reveals that the CoQ 10 H 2 /CoQ 10 ratio is substantially higher in the cytosol than in mitochondria. During ferroptosis, compartment-specific remodeling of CoQ redox status is observed, consistent with distinct roles for CoQ in plasma membrane and mitochondrial biology. The platform also enables robust analysis of plasma CoQ redox status in mouse and human samples. These advances provide a broadly applicable approach for studying redox biology, ferroptosis, mitochondrial metabolism, and biomarker discovery.