Abstract Skeletal muscle represents a physiologically relevant model for the application of redox proteomic techniques to dissect its response to exercise and aging. Contracting skeletal muscles generate ROS (reactive oxygen species) and RNS (reactive nitrogen species) necessary for the regulation of many proteins involved in excitation-contraction coupling. The magnitude and species of ROS/RNS generated by contracting muscles will have downstream effects on specific protein targets and cellular redox signalling. Redox modifications on specific proteins are essential for the adaptive response to exercise and skeletal muscle can develop a dysregulated redox response during aging. In the present article, we discuss how redox proteomics can be applied to identify and quantify the reversible modifications on susceptible cysteine residues within those redox-sensitive proteins, and the integration of oxidative and non-oxidative protein modifications in relation to the functional proteome. Skeletal muscle as a redox model Skeletal muscle represents the largest organ of the human body and comprises approximately 40 % of total body mass in humans. Over the age of 50, there is a decline in skeletal muscle mass in adults. The decline in muscle mass and associated function is referred to as sarcopenia, and can lead to a reduction in the ability to perform daily tasks and loss of independence amongst the elderly Key words: aging, cysteine modification, exercise, redox proteomics, signalling, skeletal muscle. Abbreviations: eNOS, endothelial NOS; ICAT, isotope-coded affinity tag; iTRAQ, isobaric tag for relative and absolute quantification; MRM, multiple reaction monitoring; NOS, nitric oxide synthase; PTM, post-translational modification; RNS, reactive nitrogen species; ROS, reactive oxygen species. 1 To whom correspondence should be addressed (email [email protected]). A number of questions remain as to the source of the signals required for the generation of ROS and RNS, the identification of their specific protein targets (and modifications) and how the redox signals are relayed throughout the muscle fibre. The intensity of muscle contractions, muscle fibre type, and the fitness and the age of the individual may all have an effect on both the levels and type of ROS/RNS generated. Aging muscle has an altered redox response with subsequent physiological and biochemical effects on the cytoskeleton, mitochondria, Ca 2 + signalling and sequestration. Exercise is known to induce the generation of ROS and RNS that results in the activation of a number of transcription factors, including NF-κB (nuclear factor κB), AP-1 (activator protein 1) and HSF-1 (heat-shock factor 1), and induces mitochondrial biogenesis (for a review, see Defining the biochemical pathways and processes within skeletal muscles that are affected by ROS/RNS and how these responses change with age and exercise could help our understanding of biological aging. Moderate levels of ROS/RNS generally act through a reversible thiol-disulfideexchange mechanism on specific cysteine residues and can modify key target proteins by altering both the structur
