Proximity labeling is a powerful approach for detecting protein-protein interactions. Most proximity labeling techniques use a promiscuous biotin ligase or a peroxidase fused to a protein of interest, enabling the covalent biotin labeling of proteins and subsequent capture and identification of interacting and neighboring proteins without the need for the protein complex to remain intact. To date, only a few studies have reported on the use of proximity labeling in plants. Here, we present the results of a systematic study applying a variety of biotin-based proximity labeling approaches in several plant systems using various conditions and bait proteins. We show that TurboID is the most promiscuous variant in several plant model systems and establish protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions. We demonstrate the applicability of TurboID in capturing membrane-associated protein interactomes using Lotus japonicus symbiotically active receptor kinases as a test case. We further benchmark the efficiency of various promiscuous biotin ligases in comparison with one-step affinity purification approaches. We identified both known and novel interactors of the endocytic TPLATE complex. We furthermore present a straightforward strategy to identify both nonbiotinylated and biotinylated peptides in a single experimental setup. Finally, we provide initial evidence that our approach has the potential to suggest structural information of protein complexes.

邻近标记是检测蛋白质-蛋白质相互作用的有效方法。大多数邻近标记技术使用混杂的生物素连接酶或与目标蛋白质融合的过氧化物酶，从而能够对蛋白质进行共价生物素标记，并随后捕获和识别相互作用的蛋白质和邻近蛋白质，而不需要蛋白质复合物保持完整。迄今为止，只有少数研究报告了邻近标签在植物中的使用。在这里，我们展示了一项系统研究的结果，该研究在使用不同条件和诱饵蛋白的几种植物系统中应用了各种基于生物素的邻近标记方法。我们证明 TurboID 是几种植物模型系统中最混杂的变体，并建立了将基于质谱的分析与苛刻的提取和洗涤条件相结合的协议。我们使用莲花共生活性受体激酶作为测试案例，证明了 TurboID 在捕获膜相关蛋白相互作用组方面的适用性。我们进一步对各种混杂生物素连接酶的效率与一步亲和纯化方法进行比较。我们鉴定了内吞 TPLATE 复合物的已知和新型相互作用因子。此外，我们还提出了一种简单的策略，可以在单个实验设置中识别非生物素化和生物素化的肽。最后，我们提供了初步证据，表明我们的方法有可能提供蛋白质复合物的结构信息。