Guo Hongyang, Zhang Chengxue, Qin Wei
School of Pharmaceutical Sciences, Tsinghua University, Beijing, China.
Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China.
Methods Mol Biol. 2025;2953:243-260. doi: 10.1007/978-1-0716-4694-6_16.
Protein functions are tightly associated with their locations and can be dynamically regulated by movement between cellular regions. Traditional methods, such as fluorescent protein-based imaging and fractionation-coupled proteomics, have fundamental limitations in the unbiased discovery of protein trafficking events. Proximity labeling (PL) technology has emerged as a versatile tool in spatial proteomics based on genetically encoded promiscuous enzymes, which generate reactive species to tag endogenous proteins. Although it has been widely applied to provide snapshots of subcellular proteomes, traditional PL fails to effectively map dynamic protein trafficking. To overcome this challenge, TransitID was developed to map endogenous proteome trafficking with nanometer spatial resolution within and between living cells. Two orthogonal PL enzymes, TurboID and APEX2, are targeted to source and destination regions, respectively, followed by tandem PL to record protein movement. Here, we summarize the materials and experimental procedures for TransitID, enabling the analysis of protein trafficking between organelles and neighboring cells.
蛋白质功能与其定位紧密相关，并可通过细胞区域间的移动进行动态调控。传统方法，如基于荧光蛋白的成像技术和分级分离耦合蛋白质组学，在无偏发现蛋白质转运事件方面存在根本局限性。邻近标记（PL）技术已成为基于基因编码的混杂酶的空间蛋白质组学中的一种通用工具，该酶会产生活性物质来标记内源性蛋白质。尽管它已被广泛应用于提供亚细胞蛋白质组的快照，但传统的PL无法有效绘制动态蛋白质转运图谱。为了克服这一挑战，开发了TransitID，用于在活细胞内和细胞间以纳米空间分辨率绘制内源性蛋白质组转运图谱。两种正交的PL酶TurboID和APEX2分别靶向源区域和目标区域，随后进行串联PL以记录蛋白质移动。在此，我们总结了TransitID的材料和实验步骤，可用于分析细胞器之间以及相邻细胞之间的蛋白质转运。
