FlAsH-ID enables live-cell photoproximity labeling with a short peptide tag

Proximity labeling has emerged as a valuable tool for the interrogation of protein–protein and protein–RNA interactions. Almost all proximity labeling methods are deployed using large fusion proteins, which, while powerful in many contexts, can disrupt native biology or obscure important epitopes on the protein under study. Here we develop a photoproximity labeling method that is localized to a short 6-aa peptide sequence. Termed FlAsH-ID, this method uses the combination of the Tsien tetracysteine tag with the FlAsH dye for localized singlet oxygen generation. We demonstrate that this green-light-based method is nontoxic and can be used to label proteins within several cellular compartments. Lastly, we establish that FlAsH-ID can be deployed in primary CD8+ T cells to assess the interactome of the transcription factor RUNX3. Our results suggest this technique will expand the field of proximity labeling to small, functionally sensitive proteins in primary cells without perturbing their function. Commonly used proximity labeling methods implement large fusions of a protein of interest to a proximity labeling enzyme, which can block key protein interactions and disrupt native biology. Now, a method called FlAsH-ID has been developed that enables photoproximity labeling of a protein of interest using a 6-aa peptide tag to bind a small-molecule photocatalyst.