Tunable Cell Surface Proximity Labeling via Photocatalytic and Enzymatic Activation of Fast Bioorthogonal Chemistry

Described is the use of photocatalytic and enzymatic activation of rapid bioorthogonal chemistry for cell surface proximity labeling. Current approaches to proximity labeling rely on the activation of reactive intermediates (e.g., carbenes, nitrenes, radicals, electrophiles), whose half-life is regulated by uncontrollable quenching by water and native biomolecules in the cell or media. Here, we describe an alternative proximity labeling approach based on the fastest variant of the bioorthogonal tetrazine ligation (k₂ = 3.3 × 10⁷ M^-1s^-1), where the reaction half-life can be tuned by simply controlling the concentration of a strained trans-cyclooctene (s-TCO) quencher. An electrophilic s-TCO-NHS ester is initially used to pan-label the proteome prior to activation, providing a bioorthogonal handle for tagging proteins. Subsequently, antibody-photocatalyst or antibody-enzyme conjugates are targeted to CD45 on the surface of live cells and used to locally catalyze the conversion of dihydrotetrazine molecules to reactive tetrazines. Photocatalysis is achieved through brief, 20-s irradiation at 470 nm with a commercially available antibody-fluorescein conjugate, and enzymatic catalysis is achieved using a commercially available HRP-antibody conjugate. Freely diffusing s-TCO-CO₂H serves as a quencher during the proximity labeling reaction, and adjusting quencher concentration can attenuate labeling, as demonstrated in vitro on a model protein. Tuning the half-life of the proximity labeling reaction was also studied in live cells to give proteomic data sets with high overlap. Labeling reactions with shorter half-lives enriched fewer total proteins but maintained enrichment of known interactors. We anticipate that this use of fast bioorthogonal reactions will be broadly applicable for studying protein-protein interactions.