Solid-Phase Compatible Silane-Based Cleavable Linker Enables Custom Isobaric Quantitative Chemoproteomics.

Area of Science:

• Proteomics
• Chemical Biology
• Drug Discovery

Background:

• Mass spectrometry-based chemoproteomics is vital for functional biology and drug discovery.
• Existing methods face challenges in reagent synthesis and sample preparation throughput.

Purpose of the Study:

• To establish a novel chemoproteomics workflow, silane-based cleavable isotopically labeled proteomics (sCIP), to overcome current limitations.
• To enable high-throughput, efficient sample preparation and analysis for identifying protein-ligand interactions.

Main Methods:

• Developed a scalable synthesis for dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc)-protected amino acids.
• Created customizable, isotopically labeled, and chemically cleavable biotin capture reagents.
• Implemented sCIP with MS1/MS2 quantitation, featuring click-assembled isobaric tags for early labeling and pooling, enabling six-plex multiplexing.

Main Results:

• The sCIP method streamlines sample preparation, increasing throughput and data coverage.
• Paired with FragPipe analysis, sCIP identified known and novel cysteine-ligand pairs.
• Discovered that the mitochondrial uncoupling agent FCCP functions as a covalent-reversible cysteine-reactive electrophile.

Conclusions:

• The sCIP method offers a robust and efficient platform for chemoproteomics.
• This approach advances functional biology and drug discovery by enabling comprehensive identification of cysteine-ligand interactions.
• The findings highlight FCCP's novel mechanism of action as a cysteine-reactive electrophile.