Karunadasa Sumudu S, Grismer TaraBryn S, Zhai Wenxuan, Reyes Andres V, Xu Shou-Ling
Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA.
Carnegie Mass Spectrometry Facility, Carnegie Institution for Science, Stanford, CA, USA.
Methods Mol Biol. 2025;2953:115-126. doi: 10.1007/978-1-0716-4694-6_8.
TurboID-based proximity labeling, combined with mass spectrometry (PL-MS), has been widely adopted for studies in both plants and animals for a variety of applications. In this approach, an engineered biotin ligase, TurboID, fused to a bait protein, biotinylates proteins in close proximity to the bait protein following biotin treatment, allowing their subsequent isolation using streptavidin beads and identification by mass spectrometry. This chapter provides a detailed step-by-step protocol for sample preparation using Arabidopsis tissues, along with an overview of data analysis strategies and software tools for the reliable identification and quantification of biotinylated proteins.
基于TurboID的邻近标记技术与质谱联用（PL-MS），已在植物和动物研究的多种应用中被广泛采用。在这种方法中，一种经过工程改造的生物素连接酶TurboID与诱饵蛋白融合，在生物素处理后，将与诱饵蛋白邻近的蛋白质生物素化，随后可使用链霉亲和素磁珠分离这些蛋白质，并通过质谱进行鉴定。本章提供了一份使用拟南芥组织进行样品制备的详细分步方案，以及数据分析策略和软件工具的概述，用于可靠地鉴定和定量生物素化蛋白。
