Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry
Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry
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DOI：
10.1007/978-1-4939-6747-6_10
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Gingras, Anne-Claude
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作者：
Hesketh, Geoffrey G.;Youn, Ji-Young;Gingras, Anne-Claude
要完全了解细胞功能，需要了解蛋白质相互作用网络的组成和动力学，其重要性跨越所有分子细胞生物学领域。基于质谱的蛋白质组学方法在这一过程中发挥了重要作用，亲和纯化与质谱联用（AP-MS）现在广泛用于定义相互作用景观。传统的AP-MS方法非常适合于提供关于可溶性蛋白质-蛋白质相互作用的时间方面的信息，但是在细胞裂解和AP期间维持蛋白质-蛋白质相互作用的要求意味着弱亲和力相互作用和空间信息都丢失。最近开发的一种称为BioID的方法采用与非特异性生物素连接酶BirA* 融合的诱饵蛋白的表达，该酶诱导近端蛋白的体内生物素化。将该方法与生物素亲和富集和质谱法相结合，消除了传统AP-MS方法中固有的许多溶解度和相互作用强度问题，并为蛋白质相互作用提供了无与伦比的空间背景。在这里，我们描述了并行实施的BioID和FLAG AP-MS允许同时探索蛋白质相互作用网络的空间和时间方面。
Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost. A more recently developed method called BioID employs the expression of bait proteins fused to a nonspecific biotin ligase, BirA*, that induces in vivo biotinylation of proximal proteins. Coupling this method to biotin affinity enrichment and mass spectrometry negates many of the solubility and interaction strength issues inherent in traditional AP-MS methods, and provides unparalleled spatial context for protein interactions. Here we describe the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.