ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

Proximity labeling coupled to mass spectrometry enables in situ mapping of protein-protein interactions. Here, we have developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and a newly generated mouse model to monitor the interactome of proteasomes in vivo. We demonstrate that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on proteasome activity. Analysis of proteins labeled by tagged proteasomes retrieved more than half of the known proteasome-interacting proteins in a single mass spectrometry analysis, including assembly factors, activators and ubiquitin-cycle related proteins. We optimized the protocol for processing of proximity labeled samples to minimize contamination from streptavidin and implemented Data Independent Acquisition (DIA) mass spectrometry for label-free analysis. We demonstrate the utility of our workflow by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling can be used to identify both endogenous and small molecule-induced proteasome substrates.

邻近标记（proximity labeling）技术结合质谱分析，可实现蛋白质间相互作用的原位定位分析。本研究开发了一种基于广谱生物素连接酶（promiscuous biotin ligases）标记蛋白酶体，并结合全新构建的小鼠模型的策略，以在活体中监测蛋白酶体的相互作用组（interactome）。本研究证实，生物素连接酶可整合至完全组装的蛋白酶体中，且不会对蛋白酶体活性产生负面影响。对经标记蛋白酶体标记的蛋白质进行分析，单次质谱分析即可检出超过半数已知的蛋白酶体相互作用蛋白，其中包括组装因子、激活因子以及泛素循环相关蛋白。本研究优化了邻近标记样本的处理流程，以最大程度降低链霉亲和素（streptavidin）带来的污染，并采用数据非依赖性采集（Data Independent Acquisition，DIA）质谱技术开展无标记分析。本研究通过鉴定新型蛋白酶体相互作用蛋白、绘制小鼠各器官的相互作用组图谱，以及证实邻近标记技术可用于识别内源性及小分子诱导的蛋白酶体底物，验证了本实验流程的实用性。