Abstract
Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification.
Original language | English |
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Article number | 100455 |
Pages (from-to) | 1-15 |
Number of pages | 15 |
Journal | Molecular and Cellular Proteomics |
Volume | 22 |
Issue number | 1 |
DOIs | |
Publication status | Published - 1 Jan 2023 |
Keywords
- chemical proteomics
- activity-based protein profiling
- site-specific
- inhibitor binding site identification
- phosphonate affinity handles
- Fe(III)-immobilized metal affinity chromatography
- S-transferase-pi
- Reticulon 4
- Crystal-structure
- Thioredoxin
- Mechanism
- Peptide identification
- Cysteines
- Nitrosylation
- Glutathionylation
- Oxidation