Mechanism-Guided Development of Thioacetylene Probes for Chemoproteomic Profiling of Cellular Protein Thiyl Radicals

Protein thiyl radicals are transient reactive intermediates involved in oxidative stress and enzymatic catalysis. However, their proteome-wide, site-resolved profiling in living cells remains challenging due to the lack of suitable tools. Here, we report chemoproteomic probes enabling covalent capture and site-resolved identification of protein thiyl radicals in living cells. Designed to leverage sulfur-mediated stabilization of a vinyl radical intermediate, our thioacetylene-based probes selectively capture cysteine thiyl radicals while minimizing cross-reactivity with nucleophilic thiolates and nonradical amino acid residues. In vitro validation and live-cell chemoproteomic analysis established radical-dependent labeling and site-resolved detection of protein thiyl radicals. Notably, the probes enabled site-specific mapping of thiyl radical-associated cysteine residues in live E. coli, including Cys439 of ribonucleotide reductase A (NrdA), a canonical enzymatic thiyl radical site. Expanded residue profiling further confirmed cysteine-predominant labeling and detected probe modification at established and putative glycyl radical residues in PflB, NrdD, and YjjI, suggesting that the platform may extend beyond thiyl radicals to other protein-centered radicals. Application of the probe under oxidative and nitrosative stress revealed condition-dependent changes in cellular protein radical-associated labeling, including decreased labeling at established glycyl radical residues and bidirectional changes in cysteine radical-associated labeling. Together, these findings establish thioacetylene probes as a platform for site-resolved mapping and comparative monitoring of endogenous protein-centered radicals in living cells.