Understanding how protein–protein interaction networks in the brain give rise to cognitive functions necessitates their characterization in live animals. However, tools available for this purpose require potentially disruptive genetic modifications and lack the temporal resolution necessary to track rapid changes in vivo. Here we leverage affinity-based targeting and photocatalyzed singlet oxygen generation to identify neurotransmitter receptor-proximal proteins in the live mouse brain using only small-molecule reagents and minutes of photoirradiation. Our photooxidation-driven proximity labeling for proteome identification (named PhoxID) method not only recapitulated the known interactomes of three endogenous neurotransmitter receptors (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), inhibitory γ-aminobutyric acid type A receptor and ionotropic glutamate receptor delta-2) but also uncovered age-dependent shifts, identifying NECTIN3 and IGSF3 as developmentally regulated AMPAR-proximal proteins in the cerebellum. Overall, this work establishes a flexible and generalizable platform to study receptor microenvironments in genetically intact specimens with an unprecedented temporal resolution.

了解大脑中的蛋白质-蛋白质相互作用网络如何产生认知功能，需要在活体动物中对其进行表征。然而，可用于此目的的工具需要潜在的破坏性基因修饰，并且缺乏跟踪体内快速变化所需的时间分辨率。在这里，我们利用基于亲和力的靶向和光催化的单线态氧生成，仅使用小分子试剂和几分钟的光照射来识别活小鼠大脑中的神经递质受体近端蛋白。我们用于蛋白质组鉴定的光氧化驱动的邻近标记 （称为 PhoxID） 方法不仅概括了三种内源性神经递质受体 （α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体 （AMPAR）、抑制性 γ-氨基丁酸 A 型受体和离子型谷氨酸受体 delta-2）的已知相互作用组，而且还揭示了年龄依赖性变化，将 NECTIN3 和 IGSF3 鉴定为小脑中发育调节的 AMPAR 近端蛋白。 总体而言，这项工作建立了一个灵活且可推广的平台，以前所未有的时间分辨率研究遗传完整标本中的受体微环境。