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

Area of Science:

• Chemical Biology
• Proteomics
• Drug Discovery

Background:

• Mass spectrometry-based chemoproteomics and photoaffinity labeling are key for target discovery.
• Photocrosslinked peptide-compound adducts present analytical challenges for quantitative binding site identification.

Purpose of the Study:

• To develop a method for precise identification of drug binding sites.
• To enable quantitative comparisons of compound engagement at specific protein sites.

Main Methods:

• Introduction of the Silyl Ether Enables Chemoproteomic Interaction and Target Engagement (SEE-CITE) method.
• Incorporation of a chemically cleavable photocrosslinking handle for precise labeling.
• Extension of the MSFragger algorithm for high-confidence localization scoring.

Main Results:

• SEE-CITE enables precise site-of-labeling identification.
• The method allows head-to-head comparisons of binding site engagement by diverse compounds.
• Application to kinase inhibitors delineated known sites and uncovered novel binding sites on RTN4 and COX5A.

Conclusions:

• SEE-CITE addresses analytical challenges in chemoproteomics for drug discovery.
• The method accurately identifies known and novel small-molecule binding sites.
• SEE-CITE enhances target discovery and mode of action studies in chemical biology.