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首页> 外文期刊>Current Protocols in Cell Biology >Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes
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Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes

机译：多路复用的接近生物素化与质谱法偶联，用于定义整联蛋白粘附复合物

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摘要

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 interactionnetworks through bioinformatic analyses.
机译：BioID，邻近生物素化技术，提供了一种有价值的方法来检查蛋白质复合物中发生的相互作用，这些相互作用补充了传统蛋白生化方法的蛋白质复合物。 Bioid具有对复合物的研究有益的各种优点，包括检测不溶性和瞬时蛋白质的能力。我们已经将Bioid应用于整合素附着络合物（IACS）的研究，其位于血浆膜和肌动蛋白细胞骨架之间的连接处。使用多种BioID Baits的IACS的复杂空间注释，这反过来促进了新型近端交流器的检测，并为IAC结构提供了见解。本文介绍了IAC近端蛋白的标记和亲和纯化及其通过无标记的定量质谱分析。本文还概述了识别高信N信心邻近互动者的步骤，并通过生物信息分析询问邻近互动网络的拓扑和功能相关性。

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  • 来源
    《Current Protocols in Cell Biology》 |2020年第1期|共21页
  • 作者

    Megan R. Chastney; Craig Lawless; Martin J. Humphries;

  • 作者单位

    Faculty of Biology Medicine &

    Health Wellcome Centre for Cell‐Matrix Research Manchester Academic Health Science Centre University of Manchester Michael Smith Building Oxford Road Manchester United Kingdom;

    Faculty of Biology Medicine &

    Health Wellcome Centre for Cell‐Matrix Research Manchester Academic Health Science Centre University of Manchester Michael Smith Building Oxford Road Manchester United Kingdom;

    Faculty of Biology Medicine &

    Health Wellcome Centre for Cell‐Matrix Research Manchester Academic Health Science Centre University of Manchester Michael Smith Building Oxford Road Manchester United Kingdom;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;
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