Photo-Affinity Labeling (PAL)

Photo-affinity Labeling (PAL) or Photo Cross-linking methodology is frequently used for the identification of new drug targets and probe location and structure of binding sites. 

 

 
Working Principle

This technique utilizes photo-affinity based probes and click-chemistry to covalently label and capture the targets of biologically active small-molecule. Photo-crosslinkers such as benzophenone, diazirine, and aryl azide are used which upon photo-irradiation generates highly reactive species that reacts with the adjacent molecules resulting in a direct covalent modification. 

Workflow 

The molecule is covalently attached to a photo-affinity probe that contains a pharmacophore, a photo reactive group, and a reporter tag. The small molecule probe is incubated with the protein mixture, followed by irradiation with UV light. The UV light exposure leads to the generation of chemically reactive species that covalently crosslink the photo-affinity probe to its macromolecular binding partner(s). Covalent bond formation between the probe and the protein target(s) enable the subsequent purification using isolating/reporter tags and eventually the targets are identified using mass spectrometry-based methods.

Key Advantages
  • Can be used to label low abundance and low affinity proteins
  • Can be directly linked to a specific functional assay
Points of Consideration
  • ‘Bait-Molecule’ Structure Activity Relationship (SAR) must be well defined
  • ‘Bait-Molecule’ needs to be derivatized/modified to accommodate the coupling of peptides
  • A cell-based functional activity assay should be available