Federspiel Joel D, Cristea Ileana M
Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
Methods Mol Biol. 2019;1977:115-143. doi: 10.1007/978-1-4939-9232-4_9.
Protein complexes perform key roles in nearly all aspects of biology. Identification of the composition of these complexes offers insights into how different cellular processes are carried out. The use of affinity purification coupled to mass spectrometry has become a method of choice for identifying protein-protein interactions, but has been most frequently applied to cell model systems using tagged and overexpressed bait proteins. Although valuable, this approach can create several potential artifacts due to the presence of a tag on a protein and the higher abundance of the protein of interest (bait). The isolation of endogenous proteins using antibodies raised against the proteins of interest instead of an epitope tag offers a means to examine protein interactions in any cellular or animal model system and without the caveats of overexpressed, tagged proteins. Although conceptually simple, the limited use of this approach has been primarily driven by challenges associated with finding adequate antibodies and experimental conditions for effective isolations. In this chapter, we present a protocol for the optimization of lysis conditions, antibody evaluation, affinity purification, and ultimately identification of protein complexes from endogenous immunoaffinity purifications using quantitative mass spectrometry. We also highlight the increased use of targeted mass spectrometry analyses, such as parallel reaction monitoring (PRM) for orthogonal validation of protein isolation and interactions initially identified via data-dependent mass spectrometry analyses.
蛋白质复合物在生物学的几乎所有方面都发挥着关键作用。确定这些复合物的组成有助于深入了解不同细胞过程是如何进行的。亲和纯化与质谱联用已成为鉴定蛋白质 - 蛋白质相互作用的首选方法，但最常用于使用标记和过表达诱饵蛋白的细胞模型系统。尽管这种方法很有价值，但由于蛋白质上存在标签以及目标蛋白（诱饵）丰度较高，可能会产生一些潜在的假象。使用针对目标蛋白而非表位标签产生的抗体来分离内源性蛋白质，为在任何细胞或动物模型系统中研究蛋白质相互作用提供了一种方法，且不存在过表达的标记蛋白的相关问题。虽然从概念上讲很简单，但这种方法的使用有限，主要是因为在寻找合适的抗体和有效的分离实验条件方面存在挑战。在本章中，我们介绍了一种优化裂解条件、抗体评估、亲和纯化以及最终使用定量质谱从内源性免疫亲和纯化中鉴定蛋白质复合物的方案。我们还强调了靶向质谱分析的更多应用，例如平行反应监测（PRM），用于对最初通过数据依赖型质谱分析鉴定的蛋白质分离和相互作用进行正交验证。
