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<p>For chemical probe and drug discovery campaigns, the pairing of mass spectrometry-based chemoproteomics with photoaffinity labelling has emerged as a favoured approach for target discovery and mode of action assignment. However, photocrosslinked peptide-compound adducts raise analytic challenges for quantitative binding site discovery. Here, to address these challenges, we establish the Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE) method. SEE-CITE incorporates a fully functionalized chemically cleavable photocrosslinking handle that enables precise site-of-labelling identification and head-to-head comparisons of relative binding site engagement by chemically diverse compounds. To ensure high-confidence localization of labelled residues, we extended the MSFragger algorithm of the FragPipe computational platform to report localization scores customized for photoaffinity labelling and SEE-CITE data. When applied to scout fragments and analogues of select FDA-approved kinase inhibitors, SEE-CITE delineates known drug binding sites and uncovers small-molecule binding sites that affect the protein activity of RTN4 and COX5A.</p><p></p>

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Small-molecule binding-site discovery using silyl ether-enabled chemoproteomics

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年4月27日

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  • Chau Ngo,
  • Sho Takechi,
  • Aditya Sivakumar,
  • Miranda Villanueva,
  • Fengchao Yu,
  • Andréa B. Ball,
  • Javier Rubio,
  • Elijah Biletch,
  • Nikolas R. Burton,
  • Lisa M. Boatner,
  • Phillip Kim,
  • Alexandra C. Turmon,
  • Nithesh Perumal,
  • Marc Liesa,
  • Ajit S. Divakaruni,
  • Alexey I. Nesvizhskii,
  • Keriann M. Backus

摘要

For chemical probe and drug discovery campaigns, the pairing of mass spectrometry-based chemoproteomics with photoaffinity labelling has emerged as a favoured approach for target discovery and mode of action assignment. However, photocrosslinked peptide-compound adducts raise analytic challenges for quantitative binding site discovery. Here, to address these challenges, we establish the Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE) method. SEE-CITE incorporates a fully functionalized chemically cleavable photocrosslinking handle that enables precise site-of-labelling identification and head-to-head comparisons of relative binding site engagement by chemically diverse compounds. To ensure high-confidence localization of labelled residues, we extended the MSFragger algorithm of the FragPipe computational platform to report localization scores customized for photoaffinity labelling and SEE-CITE data. When applied to scout fragments and analogues of select FDA-approved kinase inhibitors, SEE-CITE delineates known drug binding sites and uncovers small-molecule binding sites that affect the protein activity of RTN4 and COX5A.