Chemoproteomics Reveals Unexpected Lysine/Arginine-Specific Cleavage of Peptide Chains as a Potential Protein Degradation Machinery.

Abstract

Proteins can undergo oxidative cleavage by in vitro metal-catalyzed oxidation (MCO) in either the α-amidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal α-ketoacyl products and >1000 unexpected N-terminal formyl products. Strikingly, such cleavages predominantly occur at the carboxyl side of lysine (K) and arginine (R) residues across native proteomes in situ, while MCO-induced oxidative cleavages randomly distribute on peptide/protein sequences in vitro. Furthermore, ionizing radiation-induced reactive oxygen species (ROS) also generate random oxidative cleavages in situ. These findings suggest that the endogenous formation of N-formyl and N-α-ketoacyl degradants in biological systems is more likely regulated by a previously unknown mechanism with a trypsin-like specificity, rather than the random oxidative damage as previously thought. More generally, our study highlights the utility of quantitative chemoproteomics in combination with unrestricted search tools as a viable strategy to discover unexpected chemical modifications of proteins labeled with active-based probes.