Photoaffinity Labelling

Photoaffinity labelling (PAL) is a powerful technique used to study molecular interactions between a drug and its target protein. By incorporating a photoreactive chemical group into a drug, this method allows for the covalent capture of protein-drug interactions, including transient and low-affinity interactions, enabling the identification of binding partners during drug discovery and mapping drug binding sites on the target protein. 

How it works

1. Design and synthesis: a photoactivatable group (e.g., aryl azide, diazirine) is integrated into a drug.
2. Binding: the drug is allowed to interact non-covalently with its target protein under physiological conditions.
3. Activation: upon exposure to UV light, the photoactivatable group forms a highly reactive species.
4. Crosslinking: this reactive species forms a covalent bond with nearby amino acid residues on the protein, “freezing” the interaction in place.
5. Analysis: the covalently labeled protein is analyzed using mass spectrometry (MS).
6. Labelling site identification: the labelled protein is digested into peptides and the labelling sites are identified by MS analysis and subsequent peptide mapping

Figure 1. Mechanism of photoaffinity labelling

Why choose us

At Domainex, we offer full-service support to accelerate your programmes. Our chemistry team is experienced in probe design and synthesis. We can design an appropriate photoaffinity probe for you, taking into consideration the vector for growth from your bioactive motif, the nature of the linker, the most suitable photoactivatable group and, where desired, incorporation of a reporter group for pull down experiments. Domainex chemists are familiar with handling potentially photosensitive material, and late-stage modifications to install these moieties. 

Figure 2. Representative photoactivatable probes

Once synthesised, the probes are passed seamlessly to our bioanalytical team to fine tune crosslinking conditions, such as UV wavelength, UV irradiation time, drug/protein concentration, etc. Equipped with a high-resolution Q-ToF instrument with high resolution and high mass accuracy, our intact LC-MS analysis can detect photoaffinity labelled proteins with high sensitivity. By digesting the labelled protein into peptides and performing LC-MS/MS analysis, our peptide mapping software can identify the drug binding sites with high precision.