Pardo Mercedes, Bode Daniel, Yu Lu, Choudhary Jyoti S
Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute;
Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute.
J Vis Exp. 2017 Apr 1(122):55498. doi: 10.3791/55498.
Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.
大多数蛋白质与其他蛋白质协同发挥作用；因此，为了全面理解生物过程，表征这些功能单元至关重要。亲和纯化与质谱联用（AP-MS）已成为鉴定蛋白质-蛋白质相互作用的首选方法。然而，传统的AP-MS研究提供了蛋白质相互作用的信息，但组织信息却丢失了。为了解决这个问题，我们开发了一种策略，通过在亲和纯化的蛋白质复合物进行质谱表征之前对其进行分离，来揭示蛋白质可能参与的不同功能组装。使用表位标签通过诱饵蛋白的亲和纯化和竞争性洗脱分离得到的蛋白质复合物，通过蓝色非变性电泳进行分离。使用定量质谱通过相关性分析比较蛋白质迁移图谱，可将相互作用的蛋白质分配到不同的分子实体。这种方法能够分离分子量相近的蛋白质复合物，而这些复合物可能无法通过传统的色谱技术（如凝胶过滤）分离。只需比传统的AP-geLC-MS/MS多做一点工作，我们就证明了这种策略在许多情况下可能足以在鉴定蛋白质相互作用的同时获得蛋白质复合物的拓扑信息。
