2. Academic Validation
  3. Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles

Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles

  • Mol Cell Proteomics. 2023 Jan;22(1):100455. doi: 10.1016/j.mcpro.2022.100455.
Wouter van Bergen 1,  Johannes F Hevler 1,  Wei Wu 2,  Marc P Baggelaar 3,  Albert J R Heck 4
Affiliations

Affiliations

  • 1 Biomolecular 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.
  • 2 Biomolecular 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; Singapore Immunology Network (SigN), Agency for Science, Technology, and Research (A∗STAR), Singapore, Singapore; Department of Pharmacy, National University of Singapore, Singapore, Singapore.
  • 3 Biomolecular 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. Electronic address: m.p.baggelaar@uu.nl.
  • 4 Biomolecular 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. Electronic address: a.j.r.heck@uu.nl.
PMID: 36435334 DOI: 10.1016/j.mcpro.2022.100455
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.

Keywords

Fe(III)-immobilized metal affinity chromatography; activity-based protein profiling; chemical proteomics; inhibitor binding site identification; phosphonate affinity handles; site-specific.

Figures
Products
嗨！很高兴为您提供帮助！

尊敬的 MCE 客户您好，
请您选择所在区域，我们将转接对应客服为您服务！

热门区域

A

B

C

F

G

H

J

L

N

Q

S

T

X

Y

Z

热 A B C F G H J L N Q S T X Y Z