Profiling Ligand-Induced Changes in Nuclear Localization Using Proximity Labeling-Coupled Chemoproteomics

The nuclear proteome encompasses diverse proteins that regulate critical cellular functions, including histone modifications, chromatin structure, and transcription. Mutations to many of these nuclear proteins correlate with the onset of diseases such as cancer. Due to the disease relevance of nuclear proteins, drug development efforts have focused on identifying small-molecule modulators of nuclear protein function. Covalent ligands provide a promising strategy to therapeutically target nuclear proteins that lack distinct substrate binding pockets. In particular, chemoproteomic strategies have enabled the identification of ligandable sites within the proteome, with a particular emphasis on covalent targeting of cysteine residues. Nuclear proteins are typically poorly represented in chemoproteomic workflows that utilize whole-cell lysates due to the low abundance of these proteins and the localization of nuclear proteins in multiple cellular compartments. To specifically focus on the nuclear proteome, we coupled proximity labeling using a histone-TurboID construct with chemoproteomics. Notably, this platform can be utilized to identify ligandable sites within the nuclear proteome, and monitor changes in nuclear localization and chromatin association upon exposure to covalent ligands. Here, we describe the steps required to generate histone-TurboID expressing cell lines, and apply tandem mass tag (TMT)-based quantitative proteomics to monitor protein localization changes induced by covalent ligands. Together, this methodology provides a streamlined approach toward identifying covalent ligands that regulate nuclear protein function.