115 - Enrichment of oxidised peptides of oxidised allows identification of oxidized cysteine residues in signaling proteins in vivo

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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 signaling proteins subjected to ox-PTM in vivo. However, the low sensitivity remains a major hurdle to the detection of the modified residue(s) for the low abundant less reactive proteins. In most cases, identification of the Cys that undergo oxidative modification is only achieved using recombinant proteins oxidized in vitro. Here, we report a modified method of ox-PTM protein labeling coupled to M/S that highly improved the enrichment of oxidized peptides and the identification of specific ox-PTM Cys residues in vivo. In cell lines treated with diamide, a thiol-oxidizing agent, or HOCl, we identified 2699 peptides, covering 1473 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, the use of an improved method with higher sensitivity for the detection of ox-PTM Cys residues in vivo allowed us to deepen our understanding of redox signaling. Network analyses allowed us to highlight signaling pathways enriched in oxidized proteins related to innate immunity, antiviral response and inflammation.