Mitochondria are complex and dynamic organelles that are essential to the survival of nearly every eukaryotic cell. To generate a foundation for systematic investigations of mitochondrial function and adaptation, we recently established a protein compendium of these organelles across a wide range of tissues from healthy mice. This resource, termed MitoCarta, provides a robust, yet static view of the mitochondrial proteome. We are now applying MitoCarta as a framework for quantifying how mitochondrial proteins and post-translational modifications (PTMs, e.g., phsophorylation and acetylation) change during acute and chronic metabolic perturbations, and to elucidate the role of these changes in regulating mitochondrial activity. To do so, we blend state-of-the art multi-plexed mass spectrometry-based proteomics with focused biochemistry and molecular biology approaches.In particular, we have recently taken this approach to capture the mitochondrial proteome dynamics during fasting, the onset of obesity, aging, caloric restriction and acute iron deprivation. Our analyses have revealed hundreds of dynamic phosphorylation and acetylation events and have produced quantitative, searchable maps of mitochondrial alterations across a spectrum of metabolic states. We have leveraged these data to demonstrate that key steps in ketogenesis, the TCA cycle, branched-chain amino acid degradation and fatty acid oxidation are regulated by reversible PTMs, and that the mitochondrial oxidative phosphorylation machinery is highly calibrated to cellular iron content. Moving forward, we plan to further elucidate the mitochondrial signaling network by identifying the regulatory enzymes (e.g., kinases, acetyltransferases, etc.) responsible for managing mitochondrial PTMs, and to define the functions of uncharacterized mitochondrial proteins mutated in human disease.