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Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles.

Wouter van Bergen1, Johannes F Hevler1, Wei Wu2

  • 1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands; Netherlands Proteomics Center, Utrecht, The Netherlands.

Molecular & Cellular Proteomics : MCP
|November 26, 2022
PubMed
Summary

We developed PhosID-ABPP, a novel method for precisely identifying drug binding sites on proteins. This technique efficiently enriches peptides, revealing over 500 unique drug-protein interactions and offering advantages over existing activity-based protein profiling strategies.

Keywords:
Fe(III)-immobilized metal affinity chromatographyactivity-based protein profilingchemical proteomicsinhibitor binding site identificationphosphonate affinity handlessite-specific

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Area of Science:

  • Chemical Biology
  • Proteomics
  • Drug Discovery

Background:

  • Proteins are the primary targets for most drug molecules.
  • Identifying precise drug binding sites is crucial for understanding drug mechanisms and predicting effects on protein structure and function.

Purpose of the Study:

  • To develop a method for efficient and selective enrichment of peptides bound to activity-based probes.
  • To enable the identification of exact drug binding sites on proteins.

Main Methods:

  • Developed PhosID-ABPP (activity-based protein profiling) utilizing immobilized metal-affinity chromatography-enrichable phosphonate affinity tags.
  • Applied PhosID-ABPP to identify binding sites of an alkynylated afatinib derivative (PF-06672131).
  • Investigated quantitative differences in binding sites in intact cells versus cell lysates and employed an alternative protease digestion approach.

Main Results:

  • Successfully identified over 500 unique drug binding sites reproducibly using PhosID-ABPP.
  • Quantified substantial differences in approachable binding sites between intact cells and cell lysates.
  • Captured a previously reported binding site on epidermal growth factor receptor that was elusive with trypsin alone.

Conclusions:

  • PhosID-ABPP is a powerful tool for identifying exact drug binding sites.
  • This method is highly complementary to biotin-based enrichment strategies in activity-based protein profiling.
  • PhosID-ABPP offers the advantage of direct identification of activity-based probe interaction sites.