Rick A. Homan; John D. Lapek; Christina M. Woo; Sherry Niessen; Lyn H. Jones; Christopher G. Parker
Nat. Rev. Methods Primers, 2024, 4, 30
https://doi.org/10.1038/s43586-024-00308-4

Abstract

Small molecules can serve as leads for new therapeutics as well as powerful tools to investigate biological processes. Understanding the interactions of these molecules, particularly in native biological environments, is fundamentally critical to their utility. Photoaffinity labelling (PAL) represents one of the few strategies that enable the direct mapping of interactions of small molecules with proteins. PAL uses latent functional groups that form reactive intermediates only upon exposure to light of specific wavelengths that, subsequently, covalently adduct to proximal biomolecules, allowing for their enrichment and identification. Although the original applications of PAL date to six decades ago, the more recent integration with powerful mass spectrometry-based proteomic methods has profoundly impacted the ability to illuminate molecular interactions on a global scale. In this Primer, we discuss the current state-of-the-art of PAL-based strategies for studying molecular interactions in native systems, with a focus on investigations of small molecule–protein interactions. We cover topics including the basic principles of PAL chemistries, PAL probe design, experimental considerations, data analysis and applications of PAL illustrated by recent examples. Finally, we provide our perspective on persistent challenges and our outlook on the field.