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

Hanqing Zhang; Wenjie Yuan; Yi Li; Chunyu Li; Yizhou Xie; Ning Wan; Haiping Hao; Hui Ye

doi:10.1016/j.jpha.2026.101605

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Hanqing Zhang, Wenjie Yuan, Yi Li, Chunyu Li, Yizhou Xie, Ning Wan, Haiping Hao, Hui Ye. DT-TRAP enables accurate target recognition via simplified dosing and low-temperature incubation[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101605

Citation:

Hanqing Zhang, Wenjie Yuan, Yi Li, Chunyu Li, Yizhou Xie, Ning Wan, Haiping Hao, Hui Ye. DT-TRAP enables accurate target recognition via simplified dosing and low-temperature incubation[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101605

Citation:

Hanqing Zhang, Wenjie Yuan, Yi Li, Chunyu Li, Yizhou Xie, Ning Wan, Haiping Hao, Hui Ye. DT-TRAP enables accurate target recognition via simplified dosing and low-temperature incubation[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101605

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

doi: 10.1016/j.jpha.2026.101605

a. State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China;
b. School of Pharmacy, China Pharmaceutical University, Nanjing, China

Funds:

This research was supported by the National Natural Science Foundation of China (Grant Nos.: 82322068 to H.Y., 82321005 to H.H., and 32301242 to N.W.), the Project of State Key Laboratory of Natural Medicines for China Pharmaceutical University (Grant Nos.: SKLNMKF202504 and SKLNMZZ202506 to H.Y., and SKLNMZZ202402 to H.H.), the Regional University–Industry Technology Transfer Center for Biopharmaceuticals (Nanjing, Jiangsu) Early-Stage Translational Grant (Grant No.: JB2025212-3 to H.Y.), the Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China (Grant No.: JYB2025XDXM601 to H.H.), the Lingang Laboratory (Grant No.: LGL-2615-04 to H.H.), and the Natural Science Foundation of Jiangsu Province (Grant No.: BK20231020 to N.W.).

Received Date: Sep. 25, 2025
Accepted Date: Mar. 09, 2026
Rev Recd Date: Mar. 09, 2026
Available Online: Mar. 12, 2026

Abstract

Abstract

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.
- Target-responsive accessibility profiling,
- Chemical proteomics,
- Drug target discovery,
- Incubation temperature,
- Rational dosing,
- Nonspecific binding events

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References(43)

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