A quantitative redox proteome of the human muscle response to exercise
Lei YuiD, Jonathan J. PetrocelliiD, Anita Reddy, Carlos Henríquez‐OlguíniD, Nils BurgeriD, Sanghee ShiniD, Bingsen ZhangiD, Taylor Covington, Markus Waldeck‐WeiermairiD, Jiuchun ZhangiD, Shelley M WeiiD, Thomas E. JenseniD, Christian T. VoldstedlundiD, Christian Strini CarliD, Bente KiensiD, Erik Arne RichteriD, Edward T. ChouchaniiD
Abstract
Reactive oxygen species (ROS) regulate protein function through reversible cysteine oxidation. In human skeletal muscle, exercise-induced ROS initiates adaptations such as mitochondrial biogenesis, increased insulin sensitivity, and hypertrophy. However, specific protein targets of ROS regulation during exercise remain unclear owing to longstanding challenges in analyzing redox proteomes in vivo. We applied cysteine derivatization and multiplexed proteomics to map muscle protein cysteine oxidation in humans during exercise. The OxiMuscle dataset quantifies reversible modifications across 9,177 unique cysteine sites on 2,782 proteins, comprising 17,492 individual cysteine site measurements in young men undergoing three types of exercise, providing the first comprehensive, site-resolved and quantitative analysis of the exercise-regulated redox cysteine proteome in humans. We systematically define cysteine oxidation targets regulated by at least one form of exercise, many of which reside in proteins with established roles in muscle physiology. Among these sites is a redox-regulated cysteine on the autophagy receptor protein p62. We demonstrate that reversible oxidation of this cysteine regulates p62-mediated autophagy upon myotube contraction and mouse muscle adaptation to exercise in vivo. Together, these results define a redox-driven mechanism linking exercise-induced autophagy to muscle adaptation. More broadly, our findings offer a comprehensive resource on redox-signaling networks in human muscle, accessible at http://oximuscle-alb-1899330623.us-east-1.elb.amazonaws.com/.
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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (17 Sept 2026) · CC BY 4.0
Site-resolved redox proteomics of human muscle after three exercise types shows modality-specific cysteine oxidation, and a p62 Cys113 switch tied to autophagy in myotubes and mitochondrial remodeling in mice.
The direct protein targets of exercise-induced ROS in human muscle were unknown, because earlier muscle redox surveys covered few pre-defined targets or used non-stoichiometric quantification. This work measures site occupancy proteome-wide with a cysteine-reactive phosphate tag plus multiplexed MS, and links one site (p62 Cys113) to autophagy and exercise adaptation.
Key results
ShownOxiMuscle quantifies reversible cysteine oxidation at large scale in human vastus lateralis. Exercise caused no bulk oxidative shift, and only a small subset of sites changed by more than 10%.
9,177 unique cysteine sites on 2,782 proteins, 17,492 site measurements, n = 8 men per condition. 697 exercise-regulated sites, 8% of the 9,177 mapped. Most cysteines unchanged in every modality. · A Quantitative Landscape of Protein Cysteine…; Population Characteristics of Redox-Regulated…
ShownRegulated cysteines were largely specific to the exercise type. Sprint exercise modified the most sites, and the overlap of highly modified sites across modalities was weak.
Of 697 dynamic exercise-specific sites: 407 in sprint, 185 in endurance, 105 in resistance. Between 2% and 7% of proteins carried at least one cysteine changed by more than 10% per modality. · Population Characteristics of Redox-Regulated…; Exercise-mediated Redox Regulation of Individual…
ShownRegulated cysteines converged on sarcomere and calcium-signaling networks shared by all exercise types. Each modality also showed its own pathway signature: glycolysis after sprint, oxidative phosphorylation after endurance, protein synthesis after resistance.
BioPlex 3.0 networks 109 and 218 were the only two with highly modified cysteines (>10%) in all three exercise conditions. Network enrichment used Fisher's exact test with Benjamini-Hochberg FDR (adjusted P < 0.05), n = 8. · Redox Regulation of Established Muscle Exercise…; Figure legends
Shownp62 Cys113 is oxidized right after sprint exercise and reverts in recovery. In C2C12 cells, the C113S substitution reduced the higher molecular weight p62 form and impaired p62/LC3B puncta and autophagosome formation, including after electrical pulse stimulation.
Other p62 cysteines did not change significantly. C113S myoblasts had fewer p62 and LC3B puncta; C113S myotubes had fewer autophagosomes at baseline and after electrical pulse stimulation. NAC added no further inhibition in C113S myoblasts. n = 3 independent replicates. · OxiMuscle Uncovers a Redox Switch that Regulates…; Figure legends
SuggestedIn knock-in mice, exercise-induced proteome remodeling differed between genotypes after voluntary wheel running. C113S muscle accumulated a distinct mitochondrial protein set, lacked the WT autophagy-factor rise, and shifted its fiber-type profile.
141 proteins rose in C113S but not WT, 52 (~37%) mitochondrial per MitoCarta 3.0. 340 rose in WT but not C113S, 132 (~38%) mitochondrial. More Type IIb and fewer Type IIx fibers in C113S; histology n = 3 mice per group, proteomics n = 9-11. · Mitochondrial Proteome Remodeling is Impaired in…; Induction of Autophagy Machinery is Blunted in…; Figure legends
Methods, data and reproducibility
Cysteine-reactive phosphate tag (CPT) labeling with IMAC enrichment and TMT multiplexing gives percent oxidation per site (oxidized channel S/N over total-cysteine channel S/N) across pre, post and 3 h recovery. Follow-up used C2C12 and p62 C113S knock-in mice with label-free proteomics.
- Vastus lateralis biopsies from untrained young men, three exercise bouts (endurance, sprint, resistance) in crossover, pre/post/3 h recovery · Homo sapiens · n = 8 participants per exercise condition · PXD083672
- C2C12 myoblasts and myotubes, WT vs CRISPR p62 C113S, with DAAO-generated H2O2, NAC, and electrical pulse stimulation · Mus musculus (C2C12 cell line) · n = 3 independent biological replicates
- Gastrocnemius label-free DIA proteomics, WT vs p62 C113S mice, sedentary vs 5-week voluntary wheel running · Mus musculus · n = 9-11 mice per group · PXD083723
- Plantaris fiber-type and cross-sectional area histology, sedentary and exercised WT vs C113S mice · Mus musculus · n = 3 mice per group
- BioPlex 3.0 interaction networks and Gene Ontology pathways used to map regulated cysteines · Homo sapiens · n = 15,650 pull-downs (BioPlex 3.0); 1423 networks
- Methods & tools
- CPT-MS (cysteine-reactive phosphate tag), TMT multiplexing with SPS-MS3, Orbitrap Eclipse with FAIMS Pro, Comet search engine (used for CPT labeling optimization searches), BioPlex 3.0, Gene Ontology, Cytoscape 3.10.4, R 4.5.1, GraphPad Prism 11.0, DIA-NN 2.3.0, timsTOF DIA-PASEF, MitoCarta 3.0
- Data & accessions
- http://oximuscle-alb-1899330623.us-east-1.elb.amazonaws.com/
- Reagents & environment
- R 4.5.1, GraphPad Prism 11.0, Cytoscape 3.10.4, DIA-NN 2.3.0.
- Reproducibility gaps
- No code repository is given; R analysis code was AI-assisted but no link is provided. Raw MS data are in PRIDE (PXD083672, PXD083723).
Limitations (6)
- The human redox landscape comes from eight young, untrained men; generalizability to women, older people and trained states is untested. · stated by the authors
- Site occupancy reports aggregate reversible oxidation and cannot distinguish sulfenylation, disulfide or S-glutathionylation at each cysteine. · stated by the authors
- The causal chain from Cys113 oxidation to autophagosome formation is inferred, and NAC has thiol-independent effects that cannot be fully excluded. · stated by the authors
- The in vivo work used one training paradigm and modest group sizes for morphology; lower-than-expected homozygote recovery and sex-dependent baseline differences suggest effects beyond exercised muscle. · stated by the authors
- Mitophagy and clearance defects are inferred from steady-state protein abundance only; no in vivo autophagic flux or mitophagy was measured, and histology used 3 mice per group. · noted in this summary
- Oxidation of p62 Cys113 was measured only in human muscle after sprint exercise; mouse muscle oxidation of this site was not reported, so the in vivo link relies on a cross-species inference. · noted in this summary
Who should read:Read this if you study exercise redox signaling or cysteine proteomics and want a stoichiometric human muscle atlas plus a p62 autophagy case study.
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