A Dimethyl-Labeling-Based Strategy for Site-Specifically Quantitative Chemical Proteomics

Area of Science:

• Chemical proteomics
• Biochemical assays
• Drug discovery

Background:

• Activity-based protein profiling (ABPP) is crucial for understanding proteome reactivity.
• Existing isotopic tandem orthogonal proteolysis (isoTOP)-ABPP methods are limited by high costs and lengthy workflows.

Purpose of the Study:

• To develop a cost-effective and efficient method for quantitative chemical proteomics.
• To enable site-specific quantification of proteome reactivity.

Main Methods:

• Combined reductive dimethyl labeling with TOP-ABPP to create the rdTOP-ABPP method.
• Utilized commercially available cleavable tags for compatibility.
• Applied rdTOP-ABPP to profile the reactivity of (1 S,3 R)-RSL3, a ferroptosis inducer.

Main Results:

• The rdTOP-ABPP method provides accurate, precise, and reproducible site-specific quantification.
• Demonstrated improved capacity for site identification and quantification compared to previous methods.
• Generated the first global, quantitative, and site-specific proteome reactivity portrait for (1 S,3 R)-RSL3.

Conclusions:

• rdTOP-ABPP is a fast, affordable, and efficient chemical proteomics strategy.
• The method enhances the ability to profile small molecule-proteome interactions with high precision.
• rdTOP-ABPP facilitates a deeper understanding of drug mechanisms and biological pathways.