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<p>We report an enantioselective protein affinity selection mass spectrometry screening approach (E-ASMS) that enables the detection of weak binders, informs on selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screen 31 human proteins against a designed library of 8,217 chiral compounds. We identify 16 binders to 12 targets, including many proteins predicted to be “challenging to ligand”, and confirm their interactions through orthogonal biophysical assays. Seven binders to six targets display enantioselective binding, with <i>K</i><sub>D</sub> values ranging from 3 to 20 µM. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) are selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanisms underlying enantioselectivity are readily explained. These results demonstrate that E-ASMS enables the identification and characterization of selective and weakly binding ligands for novel protein targets with unprecedented throughput and sensitivity.</p>

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Enantioselective protein affinity selection mass spectrometry (E-ASMS)

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月17日

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  • Xiaoyun Wang,
  • Jianxian Sun,
  • Shabbir Ahmad,
  • Diwen Yang,
  • Fengling Li,
  • U. Hang Chan,
  • Hong Zeng,
  • Conrad V. Simoben,
  • Stuart R. Green,
  • Madhushika Silva,
  • Scott Houliston,
  • Aiping Dong,
  • Albina Bolotokova,
  • Elisa Gibson,
  • Maria Kutera,
  • Pegah Ghiabi,
  • Ivan Kondratov,
  • Tetiana Matviyuk,
  • Alexander Chuprina,
  • Danai Mavridi,
  • Christopher Lenz,
  • Andreas C. Joerger,
  • Benjamin D. Brown,
  • Richard B. Heath,
  • Wyatt W. Yue,
  • Lucy K. Robbie,
  • Tyler S. Beyett,
  • Susanne Müller,
  • Stefan Knapp,
  • Dafydd R. Owen,
  • Rachel Harding,
  • Matthieu Schapira,
  • Peter J. Brown,
  • Vijayaratnam Santhakumar,
  • Suzanne Ackloo,
  • Cheryl H. Arrowsmith,
  • Aled M. Edwards,
  • Hui Peng,
  • Levon Halabelian

摘要

We report an enantioselective protein affinity selection mass spectrometry screening approach (E-ASMS) that enables the detection of weak binders, informs on selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screen 31 human proteins against a designed library of 8,217 chiral compounds. We identify 16 binders to 12 targets, including many proteins predicted to be “challenging to ligand”, and confirm their interactions through orthogonal biophysical assays. Seven binders to six targets display enantioselective binding, with KD values ranging from 3 to 20 µM. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) are selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanisms underlying enantioselectivity are readily explained. These results demonstrate that E-ASMS enables the identification and characterization of selective and weakly binding ligands for novel protein targets with unprecedented throughput and sensitivity.