蛋白质复合物组装的精确时空调控对于细胞通过细胞内信号网络响应细胞外信号实现有意义的信息流至关重要。他们的破坏会导致疾病。尽管已经对蛋白质复合物的空间和/或时间分析进行了各种尝试,在生理条件下追踪特定蛋白质复合物的全细胞动力学仍然是一个挑战。在这里,我们描述了结合标记蛋白质的内源性表达的工作流程,细胞器标记分布指导的亚细胞分级分离,支架蛋白介导的受体复合物纯化,和靶向蛋白质组学用于全细胞尺度蛋白质复合物的时空定量。我们应用我们的方法来研究EGF依赖性ErbB受体复合物的组装动力学。在使用密度梯度离心和基于细胞器标记的细胞器分配进行分馏之后,内源性ErbB复合物在不同亚细胞分级分离中得到有效富集。通过使用靶向质谱,ErbB复杂的成分表达中等至低水平的精确量化与深度覆盖,同时在时间和亚细胞空间中。我们的结果揭示了复杂行为的复杂方案,其特征是在富含胞质溶胶的亚细胞级分中形成了具有不同分子组成的多个亚复合物,质膜,内体,或者线粒体,暗示细胞器特定的ErbB功能。值得注意的是,我们的结果首次证明,激活的ErbB受体可能通过促进细胞溶质,无受体亚复合物,由Shc1、Grb2、Arhgef5、Garem1和Lrrk1组成。这些发现强调了我们的策略作为研究蛋白质复合物时空动力学的强大工具的潜力。
Precise spatiotemporal regulation of protein complex assembly is essential for cells to achieve a meaningful rely of information flow via intracellular signaling networks in response to extracellular cues, whose disruption would lead to disease. Although various attempts have been made for spatial and/or temporal analysis of protein complexes, it is still a challenge to track cell-wide dynamics of a particular protein complex under physiological conditions. Here we describe a workflow that combines endogenous expression of tagged proteins, organelle marker distribution-directed subcellular fractionation, scaffold protein-mediated receptor complex purification, and targeted proteomics for spatiotemporal quantification of protein complexes in whole cell scale. We applied our method to investigate the assembly kinetics of EGF-dependent ErbB receptor complexes. After fractionation using the density gradient centrifugation and organelle assignment based on organelle markers, endogenous ErbB complex in different subcellular fractionation was efficiently enriched. By using targeted mass spectrometry, ErbB complex components that expressed medium to low level was precisely quantified with in-depth coverage, simultaneously in time and subcellular spaces. Our results revealed a sophisticated scheme of complex behaviors characterized by multiple subcomplexes with distinct molecular composition formed across subcellular fractions enriched with cytosol, plasma membrane, endosome, or mitochondria, implying organelle-specific ErbB functions. Remarkably, our results demonstrated for the first time that activated ErbB receptors might increase their signaling range through promoting a cytosolic, receptor-free subcomplex, consisting of Shc1, Grb2, Arhgef5, Garem1, and Lrrk1. These findings emphasize the potential of our strategy as a powerful tool to study spatiotemporal dynamics of protein complexes.