Global profiling of functional histidines in live cells using small molecule photosensitizer and chemical probe relay labeling

Abstract Recent advances in chemical proteomics have focused on developing chemical probes reacting with nucleophilic amino acid residues throughout the proteome. Among the nucleophilic amino acids, histidine is an attractive candidate due to its presence in enzyme active sites, metal-binding sites, and protein-protein interaction interfaces. However, histidine has moderate nucleophilicity, and its modification is easily influenced by cysteine and lysine, resulting in poor selectivity and narrow proteome coverage. Here, we report a singlet oxygen and chemical probe relay labeling method that achieves exquisite selectivity towards histidine. A small molecular photosensitizer library is screened together with a chemical probe library to optimize the histidine labeling, enabling histidine profiling in live cells with over 7500 unique sites. We applied this method to discover unannotated histidine sites for metalloproteins and characterize the key histidine residues H309 for metabolic enzyme IDH1 and H73 for CRIP1. Finally, we used this method to probe the accessibility of histidine residues in mitophagy, revealing H138 of PARK7 as the key residue influencing the protein subcellular localization and stability. These findings demonstrate the applicability of our method in discovering functional histidine sites in the human proteome.