Chemoproteomic mapping of the N-terminal cysteinome

The oxidation status of N-terminal cysteines directly dictates protein stability via arginylation and proteasomal degradation. However, only a handful of proteins have been shown to be regulated via this pathway. To date, no methods to detect N-terminal cysteine reactivity and abundance has been reported. Here, we demonstrate, for the first time, that N-terminal cysteine targeting probes can be used in living cells to bind and quantify N-terminal cysteines, discriminating their oxidation state. Using these probes, we identify hundreds of N-terminal cysteines and show that changes in reactivity and abundance under hypoxia can be directly detected. We believe the use of these types of probes will significantly expand our knowledge of this important proteolytic pathway.