Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2021;2200:425-440. doi: 10.1007/978-1-0716-0880-7_21.
Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to the environment. However, a main limitation of traditional approaches to study protein interactions, such as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of interactions include many of the protein complexes that mediate the perception and response to cellular stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells, including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins (within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the study of proteomes in numerous organisms, its application to plant systems has only recently been realized. In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.
蛋白质组网络是生物系统的一个关键方面，介导细胞功能和对环境的反应。然而，传统的研究蛋白质相互作用的方法（如酵母双杂交和亲和纯化结合质谱分析（AP-MS））的一个主要局限性是，它们只能识别膜结合和/或不溶性蛋白质复合物的相互作用。这些类型的相互作用包括许多介导细胞刺激感知和反应的蛋白质复合物，因此具有很大的研究兴趣。邻近依赖性生物素化（PDB）与质谱分析相结合，为研究活细胞中邻近蛋白质相互作用提供了一种强大的方法，包括膜结合和不溶性复合物。PDB 方法之一，BioID，将一种混杂的生物素连接酶与感兴趣的诱饵蛋白融合，允许对邻近蛋白质进行共价生物素化（在~10nm 范围内）。可以从不需要维持蛋白质相互作用的细胞中纯化修饰后的蛋白质，然后通过质谱分析进行鉴定。虽然 BioID 已经彻底改变了许多生物体的蛋白质组学研究，但它在植物系统中的应用直到最近才得到实现。在本章中，我们概述了在模式植物拟南芥组织中进行 BioID 的方案。
