Takashima Jamie A, Woroniecka Helena A, Charest Pascale G
Department of Chemistry and Biochemistry, University of Arizona, Tucson AZ, USA.
Current address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena CA, USA.
J Biol Methods. 2023 Mar 16;10:e99010002. doi: 10.14440/jbm.2023.396. eCollection 2023.
To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in , using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in .
要全面理解任何细胞过程，我们不仅需要识别其中涉及的蛋白质，还需要了解蛋白质网络在结构和空间上是如何组织的以及随时间如何变化。然而，细胞信号通路中许多蛋白质相互作用的动态性质仍然是绘制和研究蛋白质网络的瓶颈。幸运的是，最近在哺乳动物细胞中开发的一种使用工程化抗坏血酸过氧化物酶2（APEX2）的邻近标记方法，能够在空间和时间分辨率下识别弱的和/或瞬时的蛋白质相互作用。在这里，我们以cAMP受体cAR1为例，描述了一种在[具体生物名称未给出]中成功使用APEX2邻近标记方法的方案。结合通过质谱鉴定标记的蛋白质，该方法扩展了蛋白质组学工具箱，并且应该广泛应用于识别参与[具体生物名称未给出]各种生物学过程的相互作用伙伴。
