iProXProteomicsTian LinMus Musculushttp://www.iprox.org/page/project.html?id=IPX0016748000tianlin@sysucc.org.cnSun Yat-sen University Cancer CenteriProXProximity labeling has revolutionized the study of dynamic subcellular proteomes by enabling the capture of transient protein interactions within living cells, yet the application of existing platforms to hard-to-transfect primary cells remains challenging. Here, we leverage a bioorthogonal proximity labeling platform based on the copper-dependent tyrosinase BmTyr to profile subcellular proteomes in primary T cells. This system catalyzes the subcellular incorporation of an alkyne-phenol probe, enabling subsequent click-compatible conjugation to versatile azide-bearing tags for fluorescence imaging and affinity enrichment for mass spectrometry. To expand the proximity labeling toolkit, we developed a custom azide-HiBiT/His tag mixture, which enables direct, antibody-independent validation using the same alkyne-phenol labeling chemistry, coupled with efficient elution and ultrasensitive chemiluminescent detection for low-input samples. Applying this platform to primary T cells not only validated known nuclear components of the TNFα signaling pathway but also revealed a previously unappreciated chromatin-associated localization for NKAP, providing new mechanistic insight beyond its previously described nuclear translocation. Collectively, our work establishes a powerful and flexible tool for sensitive, context-specific proteomic mapping in challenging physiological systems.A click-compatible BmTyr platform for biotin-free proximity labeling and subcellular proteome profiling in primary T cells.Zheng Xin-Nan XN, Zeng Jing-Min JM, Huang Han-Ying HY, Zhao Chuang C, Ma Sheng-Suo SS, Feng Lin L, Zhu Hao H, Tian Lin LfalseA click-compatible BmTyr platform for biotin-free proximity labeling and subcellular proteome profiling in primary T cellsProximity labeling has revolutionized the study of dynamic subcellular proteomes by enabling the capture of transient protein interactions within living cells, yet the application of existing platforms to hard-to-transfect primary cells remains challenging. Here, we leverage a bioorthogonal proximity labeling platform based on the copper-dependent tyrosinase BmTyr to profile subcellular proteomes in primary T cells. This system catalyzes the subcellular incorporation of an alkyne-phenol probe, enabling subsequent click-compatible conjugation to versatile azide-bearing tags for fluorescence imaging and affinity enrichment for mass spectrometry. To expand the proximity labeling toolkit, we developed a custom azide-HiBiT/His tag mixture, which enables direct, antibody-independent validation using the same alkyne-phenol labeling chemistry, coupled with efficient elution and ultrasensitive chemiluminescent detection for low-input samples. Applying this platform to primary T cells not only validated known nuclear components of the TNFα signaling pathway but also revealed a previously unappreciated chromatin-associated localization for NKAP, providing new mechanistic insight beyond its previously described nuclear translocation. Collectively, our work establishes a powerful and flexible tool for sensitive, context-specific proteomic mapping in challenging physiological systems.Sat May 09 00:00:00 BST 2026PXD0782051009042119177