Department of Molecular Biology, 210 Lewis Thomas Laboratory, Princeton University, Princeton, NJ, 08544, USA.
Adv Exp Med Biol. 2014;806:263-82. doi: 10.1007/978-3-319-06068-2_11.
Through an impressive range of dynamic interactions, proteins succeed to carry out the majority of functions in a cell. These temporally and spatially regulated interactions provide the means through which one single protein can perform diverse functions and modulate different cellular pathways. Understanding the identity and nature of these interactions is therefore critical for defining protein functions and their contribution to health and disease processes. Here, we provide an overview of workflows that incorporate immunoaffinity purifications and quantitative mass spectrometry (frequently abbreviated as IP-MS or AP-MS) for characterizing protein-protein interactions. We discuss experimental aspects that should be considered when optimizing the isolation of a protein complex. As the presence of nonspecific associations is a concern in these experiments, we discuss the common sources of nonspecific interactions and present label-free and metabolic labeling mass spectrometry-based methods that can help determine the specificity of interactions. The effective regulation of cellular pathways and the rapid reaction to various environmental stresses rely on the formation of stable, transient, and fast-exchanging protein-protein interactions. While determining the exact nature of an interaction remains challenging, we review cross-linking and metabolic labeling approaches that can help address this important aspect of characterizing protein interactions and macromolecular assemblies.
通过一系列令人印象深刻的动态相互作用，蛋白质成功地执行了细胞中的大多数功能。这些受时间和空间调节的相互作用提供了一种手段，使单个蛋白质能够发挥多种功能并调节不同的细胞途径。因此，了解这些相互作用的特性和本质对于定义蛋白质的功能及其对健康和疾病过程的贡献至关重要。在这里，我们提供了一个概述，介绍了包含免疫亲和纯化和定量质谱（通常缩写为 IP-MS 或 AP-MS）的工作流程，用于描述蛋白质-蛋白质相互作用。我们讨论了在优化蛋白质复合物分离时应考虑的实验方面。由于在这些实验中存在非特异性结合是一个问题，我们讨论了非特异性相互作用的常见来源，并介绍了基于无标记和代谢标记质谱的方法，这些方法可以帮助确定相互作用的特异性。细胞途径的有效调节和对各种环境压力的快速反应依赖于稳定、瞬时和快速交换的蛋白质-蛋白质相互作用的形成。虽然确定相互作用的确切性质仍然具有挑战性，但我们回顾了交联和代谢标记方法，这些方法可以帮助解决描述蛋白质相互作用和大分子组装的这一重要方面。
