Protocol for establishing a protein interactome based on close physical proximity to a target protein within live budding yeast.
Protocol for establishing a protein interactome based on close physical proximity to a target protein within live budding yeast.
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基于与活芽殖酵母内的靶蛋白的紧密物理接近性建立蛋白质相互作用组的方案。
DOI：
10.1016/j.xpro.2023.102663
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发表时间：
2023-12-15
期刊：
影响因子：
--
通讯作者：
Freeman, Brian C
中科院分区：
文献类型：
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作者：
Kolhe, Janhavi A;Babu, Neethu L;Freeman, Brian C
在这里，我们提出了一个协议，用于建立一个蛋白质相互作用组的基础上密切的物理接近活酵母细胞内的靶蛋白。我们描述了通过整合非天然氨基酸来捕获瞬时和稳定结合剂的步骤。我们详细介绍了采用定点方法标记的表面，介导的蛋白质协会和揭示的相互作用的结合位点的程序。结合质谱，我们的方法证明在发现结合伙伴和构建一个全面的蛋白质相互作用网络的价值。非天然氨基酸对苯甲酰基-L-苯丙氨酸（Bpa）是一种有效的交联剂Bpa可以选择性地掺入到目标蛋白（TP）中Bpa-TP和质谱的组合产生了详细的物理相互作用组Bpa/质谱策略识别特定的蛋白质-蛋白质相互作用位点出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。在这里，我们提出了一个协议，用于建立一个蛋白质相互作用组的基础上密切的物理接近活酵母细胞内的靶蛋白。我们描述了通过整合非天然氨基酸来捕获瞬时和稳定结合剂的步骤。我们详细介绍了采用定点方法标记的表面，介导的蛋白质协会和揭示的相互作用的结合位点的程序。结合质谱，我们的方法证明在发现结合伙伴和构建一个全面的蛋白质相互作用网络的价值。
Here, we present a protocol for establishing a protein interactome based on close physical proximity to a target protein within live yeast cells. We describe steps for capturing both transient and stable binders by integrating a non-natural amino acid. We detail procedures for employing a site-directed method for labeling the surface that mediates protein associations and uncovers the binding sites on the interactors. Combined with mass spectrometry, our approach proves valuable in discovering binding partners and constructing a comprehensive protein-interaction network. Non-natural amino acid p-benzoyl-L-phenylalanine (Bpa) is an effective cross-linker Bpa can be selectively incorporated into a target protein (TP) Combination of Bpa-TP and mass spectrometry produces a detailed physical interactome Bpa/mass spec tactic identifies specific protein-protein interaction sites Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for establishing a protein interactome based on close physical proximity to a target protein within live yeast cells. We describe steps for capturing both transient and stable binders by integrating a non-natural amino acid. We detail procedures for employing a site-directed method for labeling the surface that mediates protein associations and uncovers the binding sites on the interactors. Combined with mass spectrometry, our approach proves valuable in discovering binding partners and constructing a comprehensive protein-interaction network.