Label Free Quantitative Proteomic using Progenesis QI proteomics Reveals Novel Mechanism of Redox Regulation in Skeletal Muscles

There is a high production of reactive oxygen species in skeletal muscle during stress such as exercise. The Nrf2/Keap1 pathway plays a critical role in maintaining redox homeostasis. Information on role of Nrf2/Keap1 in skeletal muscle remains elusive. We applied Label-Free Quantitative Proteomics on Nrf2 KO and Keap1 KO novel transgenic mice models. This data was further validated using Immunoblotting, exercise capacity and soleus (Sol) muscle contractility. 50 ug of protein per sample from three biological replicates per group was taken digested with MS-grade trypsin overnight at 37 C with. Proteomic data was acquired on Thermo Orbitrap Fusion Lumos and analyzed by Progenesis QI proteomics. This data showed significant changes in Keap1 KO. More than 100 proteins (P ≤ 0.05), including Nrf2 and its targets that are involved in anti-oxidation, detoxification, anti-inflammation, and metabolism. For example, sulfiredoxin-1, immunoglobulin-K, NAD(P)H dehydrogenase (ubiquinone), carbonyl reductase, pirin, and GSTA2 were upregulated suggesting an enhancement in cytoprotective mechanisms as result of Keap1 loss. In addition to these Nrf2-targets, Keap1 KO also altered muscular structural and contractile proteins. Changes were subtle as a result of Nrf2 KO. In conclusion, Nrf2/Keap1 system seems to play a critical role in skeletal muscle contractility. The proteomics data also suggest that, at least in the Sol, the Nrf2/Keap1 pathway not only exerts cytoprotection but also modulates muscular structure and function. Keap1 KO upregulated more proteins than were downregulated by Nrf2 KO (98 vs 4), confirming the consensus that the Nrf2/Keap1 system exerts effects primarily in the stress state where Keap1 is inactivated, which can be observed during strenuous exercise and may be important in skeletal myopathies.