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Photoaffinity labelling with small molecules

Nature Reviews Methods Primers volume 4, Article number: 30 (2024) Cite this article

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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.

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Fig. 1: Interactome mapping in biological systems using photoaffinity labelling reagents.
Fig. 2: Mechanisms of photoactivation and cross-linking of various photoreactive functionalities.
Fig. 3: Experimental workflow to capture and identify interacting molecules in biological systems utilizing photoaffinity probes.
Fig. 4: Experimental controls commonly employed in photoaffinity labelling studies.
Fig. 5: Applications for photoaffinity labelling in target identification and ligandability studies.
Fig. 6: Examples of photoaffinity labelling applications to identify targets of bioactive molecules.

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Acknowledgements

C.G.P. acknowledges support from the National Institute of Allergic and Infectious Diseases (NIAID/ R01 AI156258).

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Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

    Rick A. Homan & Christopher G. Parker

  2. Belharra Therapeutics, San Diego, CA, USA

    John D. Lapek & Sherry Niessen

  3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Christina M. Woo

  4. Dana-Farber Cancer Institute, Boston, MA, USA

    Lyn H. Jones

  5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Lyn H. Jones

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  1. Rick A. Homan
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Contributions

Introduction (R.A.H., C.M.W. and C.G.P.); Experimentation (R.A.H., C.M.W., L.H.J. and C.G.P.); Results (R.A.H., J.D.L., C.M.W., L.H.J., S.N. and C.G.P.); Applications (R.A.H., L.H.J. and S.N.); Reproducibility and data deposition (R.A.H. and C.G.P.); Limitations and optimizations (R.A.H., L.H.J. and C.G.P.); Outlook (R.A.H. and C.G.P.); Overview of the Primer (C.G.P.).

Corresponding author

Correspondence to Christopher G. Parker.

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Competing interests

L.H.J. serves on the scientific advisory boards for, and holds equity in, Interline Therapeutics, Rapafusyn Pharmaceuticals, Ananke Therapeutics and Hyku Biosciences; consults for Matchpoint Therapeutics; holds equity in Jnana Therapeutics; and as the Director of the Center for Protein Degradation at DFCI receives research funding from Deerfield. The other authors declare no competing interests.

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Nature Reviews Methods Primers thanks Douglas Johnson, Euna Yoo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Comet: https://uwpr.github.io/Comet/

MaxQuant: https://maxquant.org/

MSFragger: https://msfragger.nesvilab.org/

PRIDE: https://www.ebi.ac.uk/pride/

Proteome Xchange: https://www.proteomexchange.org/

Skyline: https://www.skyline.ms/project/home/begin.view

Supplementary information

Glossary

Chemoproteomics

(Also known as chemical proteomics). An area of chemical biology focused on designing and employing chemical probes to profile small molecule–protein interactions, often through mass spectrometry-based proteomics.

Click chemistry

The ‘click’ reaction classically refers to the reaction between an azide and a terminal alkyne to form a 1,2,3-triazole, although is often used to loosely describe a set of spring-loaded chemical reactions that selectively and efficiently occur between two functional groups.

Proximity labelling

A technique used to identify and study interactions between proteins or other biomolecules in nearby space to a small molecule, protein or other biomolecule of interest. This method involves the introduction of a reactive chemical label, such as a biotin tag or photoreactive group, to catalytically react with nearby molecules within a certain spatial range.

Reductive dimethylation

(ReDiMe). A quantitation method in mass spectrometry-based proteomics wherein peptide amino termini and lysine ε-amines are covalently labelled with isotopically encoded dimethyl groups via reaction of formaldehyde (CH2O/13CD2O) and a reducing agent (for example, sodium cyanoborohydride).

Stable isotope labelling by amino acids in cell culture

(SILAC). A labelling technique used in proteomic investigations to quantify differences in protein abundance between samples, where two populations of cells are cultivated with isotopically pure heavy or light amino acids, namely lysine and arginine with either 13C or 15N in lieu of 12C and 14N.

Tandem mass tagging

(TMT). A quantitation method in mass spectrometry-based proteomics wherein peptide amino termini and lysine ε-amines are covalently labelled with an isobaric chemical tag with varying masses via N-hydroxysuccinimde ester amidation, enabling multiplexing of several conditions within a single experiment.

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Homan, R.A., Lapek, J.D., Woo, C.M. et al. Photoaffinity labelling with small molecules. Nat Rev Methods Primers 4, 30 (2024). https://doi.org/10.1038/s43586-024-00308-4

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