Protocol for clickable photoaffinity labeling and quantitative chemical proteomics

Summary Here, we describe a protocol for a photoaffinity labeling probe strategy for target deconvolution in live cells. We made a chemical probe by incorporation of a photoreactive group to covalently cross-link with adjacent amino acid residues upon UV irradiation. Click chemistry-based enrichment captures labeled proteins for proteomic analysis. Here, we detail specifics for finding targets of LXRβ, but the protocol has potential for application to other targets. For complete details on the use and execution of this protocol, please refer to Seneviratne et al. (2020).
Graphical Abstract
Highlights • Protocol detailing photoaffinity probe strategy for target deconvolution in live cells • Competition with parent compound demonstrates specific binding • Photoaffinity label provides evidence of small-molecule binding to LXRβ • Click chemistry-based enrichment captures labeled proteins for proteomic analysis
Here, we describe a protocol for a photoaffinity labeling probe strategy for target deconvolution in live cells. We made a chemical probe by incorporation of a photoreactive group to covalently cross-link with adjacent amino acid residues upon UV irradiation. Click chemistry-based enrichment captures labeled proteins for proteomic analysis. Here, we detail specifics for finding targets of LXRβ, but the protocol can be broadly applied to other targets.