The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein–protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.

细胞之间以及细胞器之间的通讯通常由膜蛋白的相互作用控制。尽管存在许多检测蛋白质相互作用的方法，但膜PPI仍然难以检测。在这里，我们开发了一种基于邻近度的标记系统PUP-IT（基于聚吡咯的相互作用标记），以识别膜蛋白相互作用。在这种方法中，将小的蛋白质标签Pup应用于与PafA融合的诱饵相互作用的蛋白质，从而使瞬态和弱相互作用得以富集并通过质谱检测。幼犬不会从酶中扩散出来，从而可以进行高特异性标记。我们将这种方法应用于CD28，CD28是T淋巴细胞活化的关键共刺激受体，并确定了已知的CD28结合伴侣和多种潜在的相互作用蛋白。