Cell Signaling and Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Methods. 2011 Aug;54(4):387-95. doi: 10.1016/j.ymeth.2011.03.001. Epub 2011 Mar 5.
Since most cellular processes depend on interactions between proteins, information about protein-protein interactions (PPIs) provide valuable insights into protein function. Over the last years, quantitative affinity purification followed by mass spectrometry (q-AP-MS) has become a powerful approach to investigate PPIs in an unbiased manner. In q-AP-MS the protein of interest is biochemically enriched together with its interaction partners. In parallel, a control experiment is performed to control for non-specific binding. Quantitative mass spectrometry is then employed to compare protein levels in both samples and to exclude non-specific contaminants. Here, we provide two detailed q-AP-MS protocols for pull-downs with immobilized bait proteins or transient transfection of tagged expression constructs. We discuss benefits and limitations of q-AP-MS and highlight critical parameters that need to be considered. The protocols and background information presented here allow the reader to adapt the generic q-AP-MS strategy for a wide range of biological questions.
由于大多数细胞过程都依赖于蛋白质之间的相互作用，因此关于蛋白质-蛋白质相互作用（PPIs）的信息为深入了解蛋白质的功能提供了有价值的见解。在过去的几年中，定量亲和纯化结合质谱（q-AP-MS）已成为一种研究蛋白质相互作用的强大方法，能够在无偏倚的情况下进行。在 q-AP-MS 中，感兴趣的蛋白质与它的相互作用伙伴一起通过生化方法进行富集。同时，还进行对照实验以控制非特异性结合。然后，采用定量质谱技术比较两个样品中的蛋白质水平，以排除非特异性污染物。在这里，我们提供了两种详细的 q-AP-MS 方案，用于固定化诱饵蛋白的下拉实验或标记表达构建体的瞬时转染。我们讨论了 q-AP-MS 的优点和局限性，并强调了需要考虑的关键参数。这里呈现的方案和背景信息使读者能够将通用的 q-AP-MS 策略应用于广泛的生物学问题。
