A click-compatible BmTyr platform for biotin-free proximity labeling and subcellular proteome profiling in primary T cells

ABSTRACT: Proximity 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.

ORGANISM(S): Mus Musculus

SUBMITTER: Tian Lin  

PROVIDER: PXD078205 | iProX | Sat May 09 00:00:00 BST 2026

REPOSITORIES: iProX