Department of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.
Adv Exp Med Biol. 2019;1158:83-100. doi: 10.1007/978-981-13-8367-0_6.
Mitochondria (mt) are double-membraned, dynamic organelles that play an essential role in a large number of cellular processes, and impairments in mt function have emerged as a causative factor for a growing number of human disorders. Given that most biological functions are driven by physical associations between proteins, the first step towards understanding mt dysfunction is to map its protein-protein interaction (PPI) network in a comprehensive and systematic fashion. While mass-spectrometry (MS) based approaches possess the high sensitivity ideal for such an endeavor, it also requires stringent biochemical purification of bait proteins to avoid detecting spurious, non-specific PPIs. Here, we outline a tagging-based affinity purification coupled with mass spectrometry (AP-MS) workflow for discovering new mt protein associations and providing novel insights into their role in mt biology and human physiology/pathology. Because AP-MS relies on the creation of proteins fused with affinity tags, we employ a versatile-affinity (VA) tag, consisting of 3× FLAG, 6 × His, and Strep III epitopes. For efficient delivery of affinity-tagged open reading frames (ORF) into mammalian cells, the VA-tag is cloned onto a specific ORF using Gateway recombinant cloning, and the resulting expression vector is stably introduced in target cells using lentiviral transduction. In this chapter, we show a functional workflow for mapping the mt interactome that includes tagging, stable transduction, selection and expansion of mammalian cell lines, mt extraction, identification of interacting protein partners by AP-MS, and lastly, computational assessment of protein complexes/PPI networks.
线粒体（mt）是双层膜的动态细胞器，在许多细胞过程中发挥着至关重要的作用，而 mt 功能的损伤已成为越来越多人类疾病的致病因素。鉴于大多数生物功能都是由蛋白质之间的物理相互作用驱动的，因此，了解 mt 功能障碍的第一步是全面系统地绘制其蛋白质-蛋白质相互作用（PPI）网络。虽然基于质谱（MS）的方法具有进行此类研究的理想高灵敏度，但它还需要对诱饵蛋白进行严格的生化纯化，以避免检测到虚假的、非特异性的 PPI。在这里，我们概述了一种基于标记的亲和纯化与质谱（AP-MS）工作流程，用于发现新的 mt 蛋白相互作用，并深入了解它们在 mt 生物学和人类生理学/病理学中的作用。由于 AP-MS 依赖于与亲和标签融合的蛋白质的创建，我们使用了一种多功能亲和（VA）标签，该标签由 3×FLAG、6×His 和 Strep III 表位组成。为了有效地将带有亲和标签的开放阅读框（ORF）递送到哺乳动物细胞中，VA 标签通过 Gateway 重组克隆克隆到特定的 ORF 上，然后使用慢病毒转导将所得表达载体稳定引入靶细胞中。在本章中，我们展示了一个映射 mt 相互作用组的功能工作流程，该流程包括标记、稳定转导、哺乳动物细胞系的选择和扩增、mt 提取、通过 AP-MS 鉴定相互作用的蛋白伴侣，以及最后对蛋白复合物/PPI 网络进行计算评估。
