A Simplified Approach for the Direct Detection of Protein Oxidation in Muscle Using In-Gel and Microplate Assays

Reactive oxygen species (ROS) are emerging as regulators of protein redox states which influence many physiological processes. Current methods for the detection of protein redox states, including mass spectrometry, are expensive and not easily accessible or poorly validated. IRdye800CW Maleimide, a highly sensitive maleimide-based infrared dye, has been reported to detect the redox state of immunoprecipitated proteins in cardiac muscle using modified western blot procedures, but has yet to be validated as a novel tool to detect redox conditions throughout the proteome using common and cost-effective assays. In this study we tested the efficacy of IRDye800CW Maleimide in detecting protein redox state in cardiac muscle using mini-gel SDS-PAGE and an in-well approach. Chemical and heat-induced modifications to cardiac tissue redox states were accurately detected with the dye in cardiac lysates. These findings were also validated against HPLC measures of GSSG content.