Reduction-Alkylation Proteome Profiling (RAPP): A novel strategy for proteome-wide target identification.

The discovery of target proteins for drugs is crucial, and mass spectrometry (MS)-based proteomics is one of the most important tools for identifying drug targets. Different target identification methodologies are suited to different applications, each offering unique strengths but also facing specific challenges. Herein, we propose Reduction-Alkylation Proteome Profiling (RAPP), a novel method for proteome-wide ligand target identification based on differential reduction-alkylation reaction. The RAPP method was successfully validated by verifying the interaction between BSA and heparin, recapitulating the known targets of staurosporine, oxamate, and entacapone, and identifying ATP-binding proteins. Beyond the expected target LDHA, RAPP also identified FAHD1 as a previously unrecognized binding target of oxamate, suggesting its potential to detect weak binding events. Furthermore, RAPP revealed proteome-wide differences in protein stability under reducing conditions. Compared with the classic TPP method, our approach enables the identification and denaturation curve fitting of 295 additional proteins that could not produce discernible melting curves in TPP, features a more simplified workflow without complicated operations or specialized equipment, and can serve as an important complementary strategy to classical modification-free ligand target identification methods. In conclusion, RAPP serves as a valuable modification-free approach for target identification and holds practical application potential in chemical biology and drug discovery research.