DT-TRAP enables accurate target recognition via simplified dosing and low-temperature incubation.

Target-responsive accessibility profiling (TRAP) is a powerful chemoproteomic strategy for drug target discovery that monitors ligand-induced changes in lysine accessibility. However, TRAP and related approaches remain constrained by high sample consumption and extended analysis time, while the systematic impact of incubation temperature on target identification remains unexplored. Here, we introduce dose- and temperature-optimized TRAP (DT-TRAP), a streamlined workflow that integrates rational dosing strategies with controlled incubation temperatures to enhance the specificity and reliability of ligand-protein interaction profiling. Using the pyruvate kinase M2 (PKM2) activator TEPP-46 and the heat shock protein 90 (HSP90) inhibitor geldanamycin as model compounds, we demonstrate that a simplified three-dose design is sufficient for the robust identification of bona fide targets. Notably, we show that incubation at 4 °C significantly suppresses nonspecific interactions and enhances binding specificity compared to 25 °C or 37 °C. Applying DT-TRAP to staurosporine and aspirin, we successfully identified multiple targets within complex proteomes, with peptide-level analyses pinpointing binding sites predominantly within ligand-binding pockets. Collectively, these findings establish DT-TRAP as a practical framework for optimizing experimental parameters in chemical proteomics, thereby improving target identification accuracy and facilitating drug discovery.