Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes.
Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes.
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DOI：
10.1002/cpcb.113
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发表时间：
2020-09-01
影响因子：
--
通讯作者：
Humphries, Martin J
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文献类型：
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作者：
Chastney, Megan R;Lawless, Craig;Humphries, Martin J
BioID 是一种邻近生物素化技术，为检查蛋白质复合物内发生的相互作用提供了一种有价值的方法，是对传统蛋白质生化方法的补充。 BioID 具有多种有利于复合物研究的优点，包括检测不溶性和瞬时蛋白质的能力。我们已将 BioID 应用于整合素粘附复合物 (IAC) 的研究，该复合物位于质膜和肌动蛋白细胞骨架之间的连接处。多个 BioID 诱饵的使用可以对 IAC 进行复杂范围的空间注释，从而促进新型近端相互作用子的检测，并提供对 IAC 架构的深入了解。本文介绍了 IAC-proximal 蛋白的标记和亲和纯化及其通过无标记定量质谱法进行的分析。本文还概述了识别高置信度邻近交互器的步骤，以及通过生物信息学分析询问邻近交互网络的拓扑和功能相关性的步骤。 © 2020 作者。基本方案 1：整合素粘附复合物成分的邻近生物素化 基本方案 2：通过 MaxQuant 进行质谱数据处理并检测高置信度的邻近相互作用子 基本方案 3：生物信息学分析和数据可视化。
BioID, a proximity biotinylation technique, offers a valuable approach to examine the interactions occurring within protein complexes that complements traditional protein biochemical methods. BioID has various advantages that are beneficial to the study of complexes, including an ability to detect insoluble and transient proteins. We have applied BioID to the study of integrin adhesion complexes (IACs), which are located at the junction between the plasma membrane and actin cytoskeleton. The use of multiple BioID baits enables a complex-wide, spatial annotation of IACs, which in turn facilitates the detection of novel proximal interactors and provides insights into IAC architecture. This article describes the labeling and affinity purification of IAC-proximal proteins and their analysis by label-free quantitative mass spectrometry. The article also outlines steps to identify high-confidence proximity interactors, and to interrogate the topology and functional relevance of proximity interaction networks through bioinformatic analyses. © 2020 The Authors. Basic Protocol 1: Proximity biotinylation of integrin adhesion complex components Basic Protocol 2: Mass spectrometry data processing by MaxQuant and detection of high-confidence proximal interactors Basic Protocol 3: Bioinformatic analysis and data visualization.