2. PROJECT SUMMARY ABSTRACT Acyl-coenzyme A thioesters (acyl-CoAs) are evolutionarily conserved critical metabolic intermediates that are made and used in distinct parts of the eukaryotic cell. In different sub-cellular compartments, acyl-CoAs can be used to meet energy needs, act as signaling molecules, and serve as acyl-donors for post-translational modifications (PTMs) of proteins. A major challenge remaining unaddressed in the field of bioanalytical chemistry is the quantitation of the subcellular localized pools of these critical metabolites, especially between the mitochondria, nucleus, and cytosol where the same metabolite can have distinct functions. We will overcome this challenge in two aims, leveraging stable isotope labeling strategies, quantitative mass spectrometry, pharmacology, toxicology, and unique cell biology models. First, we will quantify the kinetics and fate of major carbon substrates for compartmentalized acyl-CoAs in cells and heart tissue. Second, we will quantify the effect of perturbing compartment specific processes with histone deacetylase inhibitors, electron transport chain inhibitors, and genetic models on acyl-CoAs, downstream metabolites, and compartment specific PTMs. This project will allow the rigorous quantification of distinct compartments of metabolism, and thus, their quantitative study and manipulation in normal and pathological processes where metabolism goes awry.