Development of peroxidase-activated probes for protein labelling

Abstract

Proteins are vital biomolecules essential for cellular functions, including cell signaling and regulation. Protein misfolding or mislocalization can result in various diseases. Peroxidase-mediated proximity labeling has emerged as a powerful tool for studying subcellular proteome3 and protein-protein interaction. However, traditional probe (biotin-phenol) suffers from limitations including low efficacy protein enrichment, formation of oxidized and polymerized products. To address these challenges, we developed a novel probe, N-(4-amino-3,5-dimethylbenzyl) desthiobiotinamide (desMe). Western blot analysis demonstrated efficient labeling of bovine serum albumin. Liquid chromatography-mass spectrometry (LC-MS) data confirmed the formation of one-on-one adducts from in vitro labeling reaction. Notably, the novel probe also exhibited labeling activity towards DNA and RNA. Moreover, desMe also permits APEX2-mediated labeling within the mitochondrial matrix of HEK293T cells, highlighting its potential application in cellulo. This expands the scope of applications for peroxidase-mediated proximity labeling, enabling investigations into subcellular localization and protein structure with broader implications for understanding cellular processes and disease mechanisms.