Protocol to pinpoint oxidized and reduced cysteine residues in brown fat using differential alkylation labeling techniques

Cysteine post-translational modifications are important for protein functions and protein-protein interactions. Here, we present a protocol to detect the reversibly oxidized and reduced cysteine residues of proteins using differential alkylation labeling techniques with liquid chromatography-mass spectrometry (LC-MS) analysis. We describe steps for tissue sample preparation, differential alkylation, trypsin digestion, and LC-MS analysis. We then detail procedures for protein identification and data analysis. This protocol has potential application in pinpointing the modified cysteine residues in organs and cells in various disease models. For complete details on the use and execution of this protocol, please refer to Oo et al.¹.