Identification of oxidized cysteine residues in signaling proteins in vivo

Detection of oxidative post-translational modifications (ox-PTMs) of Cys residues in mammalian signaling proteins in vivo is one of the current challenges in redox biology. Ox-PTM residues labeling using maleimide switch methods coupled to mass spectrometry (M/S) or immunoblot have rendered possible the identification of ox-PTM signaling proteins in vivo. However, the low sensitivity remains a major hurdle to the detection of the modified residue(s). In most cases, identification of these modifications requires oxidation of recombinant proteins. Here, we report a modified method of ox-PTM protein labeling coupled to M/S that permits the identification of specific ox-PTM Cys residues. In cell lines treated with a thiol oxidizing agent, we identified about 1000 peptides, covering 600 proteins, containing at least 1 ox-PTM Cys residue. Previously reported ox-PTM residues were confirmed in vivo, validating our approach. Signaling proteins, including kinases, phosphatases and transcription and translation factors are largely represented in proteins containing ox-PTM Cys. Altogether we report a method with an improved sensitivity allowing detection of ox-PTM Cys residues in vivo. We believe this method will allow deepening the understanding of redox signaling.