A disubstituted aniline probe for enhanced peroxidase-based proximal protein labelling

Abstract

Proteins are biomolecules essential for cellular functions, including cell signaling and regulation. Protein misfolding or mislocalisation can result in various diseases. Peroxidase-mediated proximity labelling has emerged as a powerful tool for studying subcellular proteome and protein–protein interactions. However, the traditional probe, biotin-phenol, suffers from limitations including low protein enrichment efficiency, and the formation of oxidised and polymerised products, complicating the downstream analysis. To address these challenges, a novel probe, N-(4-amino-3,5-dimethylbenzyl)desthiobiotinamide (DBA-Me), for protein labelling in living cells was developed. Western blot analysis demonstrated efficient labelling of bovine serum albumin in vitro. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data confirmed the formation of one-to-one adducts from the in vitro labelling reaction. Notably, this novel probe (DBA-Me) also exhibited labelling activity towards nucleic acids. Moreover, DBA-Me also permits APEX2-mediated labelling within the mitochondrial matrix of HEK293FT cells, and demonstrated improved recovery of labelled proteins after streptavidin enrichment compared to the conventional biotin-phenol (BP) probe, highlighting its superior potential application in cellulo. This facilitates peroxidase-mediated proximity labelling applications in subcellular localisation of proteins, and protein structures, with broader implications for understanding cellular processes and disease mechanisms.