Phototriggered profiling of receptor-proximal proteins in vivo in minutes

Neurotransmitter receptors are regulated by an extensive and dynamic network of protein-protein interactions. Understanding how these networks control synaptic transmission and give rise to higher-order brain functions necessitates their investigation in the live mammalian brain. However, tools available for this purpose lack the temporal resolution necessary to capture rapid changes in the interactome in live animals and require potentially disruptive genetic modifications to the animal under study. Here, we describe a method for light-activated proximity labelling in the live mouse brain that relies solely on small-molecule reagents and achieves a minute-order temporal resolution. Named PhoxID (photooxidation-driven proximity labelling for protein identification), this method involves selectively tethering a chemical photosensitiser to neurotransmitter receptors of interest and enabled us to identify characteristic as well as less studied interactors of the endogenous -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and the {psi}-aminobutyric acid type A receptor (GABAAR) with just minutes of in-brain green light irradiation. Furthermore, PhoxIDs temporal precision allowed us to capture molecular snapshots of the AMPAR-proximal proteome in the postnatal developing cerebellum, leading to the discovery of age-dependent shifts. Overall, this work establishes a highly flexible and generalisable platform to study receptor interactomes and proximal microenvironments in genetically intact specimens with an unprecedented temporal resolution.