Liu Chen, Moschou Panagiotis N
State Key Laboratory of Biocontrol, Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China.
Department of Biology, University of Crete, Heraklion, Greece.
Methods Mol Biol. 2025;2953:143-166. doi: 10.1007/978-1-0716-4694-6_10.
Capturing interactions in transient protein assemblies is a challenging task. We introduce an approach named APEAL (Tandemly Coupled Affinity Purification with Proximity-dEpendent LigAtion) for determining both transient and stable protein assemblies in Arabidopsis and other plants. APEAL exploits TurboID, a biotin ligase variant that operates efficiently at "plant-friendly" temperatures. The APEAL method begins by enriching strong or long-lived protein-protein interactions through an affinity purification step. It then captures weaker and more transient interactions through a proximity-dependent ligation step. At both stages, samples can be analyzed using immunoblotting or mass spectrometry to extract relevant data. This chapter details the APEAL procedure from construct design and pilot experiments to condition testing, proteomic sample collection, and mass spectrometry data analyses.
捕捉瞬时蛋白质组装体中的相互作用是一项具有挑战性的任务。我们介绍了一种名为APPEAL（基于邻近依赖性连接的串联偶联亲和纯化）的方法，用于确定拟南芥和其他植物中的瞬时和稳定蛋白质组装体。APPEAL利用TurboID，一种在“植物友好”温度下高效运作的生物素连接酶变体。APPEAL方法首先通过亲和纯化步骤富集强或长寿命的蛋白质-蛋白质相互作用。然后通过邻近依赖性连接步骤捕捉较弱和更瞬时的相互作用。在两个阶段，都可以使用免疫印迹或质谱分析样品以提取相关数据。本章详细介绍了从构建体设计和预实验到条件测试、蛋白质组学样品收集和质谱数据分析的APPEAL程序。
