Cai, D.; Zou, K.; Wang, J.; Zhu, H.; Ma, Y.; Yang, D.; Zhang, X.; Yan, J.; Zou, L.; Wang, K.; Ye, M.

Accurate identification of drug target proteins remain major challenges in proteomics-based target discovery, particularly for low-abundance nuclear proteins that are difficult to detect because of the complexity of whole-cell lysates. Here, we developed a detergent-free nuclear-cytoplasmic fractionation strategy compatible with peptide-centric local stability analysis (PELSA), which markedly improves detection of nuclear drug targets. Using K562 cells, we demonstrated that mild detergent-free fractionation enables high-fidelity nuclear-cytoplasmic separation with minimal cross-contamination. When coupled with PELSA, this workflow significantly increases the number of detected nuclear targets relative to whole-cell analysis. Benchmarking with well-characterized nuclear drugs, including the histone deacetylase inhibitor panobinostat and the RNA polymerase II inhibitor -amanitin, our results showed improved identification of canonical nuclear targets. Broad profiling of staurosporine target further revealed expanded kinase target coverage by combining the results of nuclear and cytoplasmic fraction, with the CLK family kinases detected exclusively in the nuclear fractions. Additionally, we showed that PELSA can also be performed on intact nucleus level. Collectively, these findings establish detergent-free nuclear-cytoplasmic fractionation-PELSA as a robust and scalable strategy for spatially resolved drug target identification, improving sensitivity for nuclear and low-abundance proteins.