Highlights d Comprehensive quantification of respiratory subunits within supercomplexes d Complexes II and V assemble within supercomplexes d Mitochondrial-encoded subunits display elevated upregulation upon exercise training d Exercise increases ubiquinone biosynthesis enzyme and decreases Lactb complexes

Mitochondrial respiratory complex subunits assemble in supercomplexes. Studies of 13 supercomplexes have typically relied upon antibody-based protein quantification, often 14 limited to the analysis of a single subunit per respiratory complex. To provide a deeper 15 insight into mitochondrial and supercomplex plasticity, we combined Blue Native 16Polyacrylamide Gel Electrophoresis (BN-PAGE) and mass spectrometry to determine the 17 supercomplexome of skeletal muscle from sedentary and exercise-trained mice. We 18 quantified 422 mitochondrial proteins within ten supercomplex bands, in which we showed 19 the debated presence of complex II and V. Upon exercise-induced mitochondrial biogenesis, 20 non-stoichiometric changes in subunits and incorporation into supercomplexes was 21 apparent. We uncovered the dynamics of supercomplex-related assembly proteins and 22 mtDNA-encoded subunits within supercomplexes, as well as the complexes of ubiquinone 23 biosynthesis enzymes and Lactb, a mitochondrial-localized protein implicated in obesity. 24Our approach can be applied to broad biological systems. In this instance, comprehensively 25 analyzing respiratory supercomplexes illuminates previously undetectable complexity in 26 mitochondrial plasticity. 27 28 Highlights: 29 • Comprehensive quantification of respiratory subunits within supercomplexes 30 • Complex II and V assemble within supercomplexes 31 • Mitochondrial-encoded subunits display elevated upregulation upon exercise training 32 2 • Exercise increases ubiquinone biosynthesis enzyme complexes 33 34

Acute exercise and chronic exercise training elicit beneficial whole‐body changes in physiology that ultimately depend on profound alterations to the dynamics of tissue‐specific proteins. Since the work accomplished during exercise owes predominantly to skeletal muscle, it has received the majority of interest from exercise scientists that attempt to unravel adaptive mechanisms accounting for salutary metabolic effects and performance improvements that arise from training. Contemporary scientists are also beginning to use mass spectrometry‐based proteomics, which is emerging as a powerful approach to interrogate the muscle protein signature in a more comprehensive manner. Collectively, these technologies facilitate the analysis of skeletal muscle protein dynamics from several viewpoints, including changes to intracellular proteins (expression proteomics), secreted proteins (secretomics), post‐translational modifications as well as fiber‐, cell‐, and organelle‐specific changes. This review aims to highlight recent literature that has leveraged new workflows and advances in mass spectrometry‐based proteomics to further our understanding of training‐related changes in skeletal muscle. We call attention to untapped areas in skeletal muscle proteomics research relating to exercise training and metabolism, as well as basic points of contention when applying mass spectrometry‐based analyses, particularly in the study of human biology. We further encourage researchers to couple the hypothesis‐generating and descriptive nature of omics data with functional analyses that propel our understanding of the complex adaptive responses in skeletal muscle that occur with acute and chronic exercise.