Wang Xiaorong, Huang Lan
Department of Physiology & Biophysics, University of California, Irvine, Medical Science I, D233, 160 Aldrich Hall, Irvine, CA, 92697, USA.
Methods Mol Biol. 2014;1188:191-205. doi: 10.1007/978-1-4939-1142-4_14.
Protein-protein interactions are essential to various physiological processes in living cells. A full characterization of protein interactions is critical to our understanding of their roles in the regulation of protein functions. Affinity purification coupled with mass spectrometry (AP-MS) has become one of the most effective approaches to systematically study protein-protein interactions. In combination with quantitative mass spectrometry, specific interacting proteins can be efficiently distinguished from nonspecific background proteins. Based on interaction affinity and kinetics, protein interactions can be classified into different categories such as stable and dynamic interactions. Standard biochemical methods are effective in capturing and identifying stable protein interactions but are not sufficient enough to identify dynamic interactors. In this chapter, we describe integrated strategies to allow the identification of dynamic interactors of protein complexes by incorporating new sample preparation methods with SILAC-based quantitation.
蛋白质-蛋白质相互作用对于活细胞中的各种生理过程至关重要。全面表征蛋白质相互作用对于我们理解其在蛋白质功能调节中的作用至关重要。亲和纯化结合质谱分析（AP-MS）已成为系统研究蛋白质-蛋白质相互作用最有效的方法之一。结合定量质谱分析，可以有效地将特异性相互作用蛋白与非特异性背景蛋白区分开来。基于相互作用亲和力和动力学，蛋白质相互作用可分为不同类别，如稳定相互作用和动态相互作用。标准生化方法在捕获和鉴定稳定蛋白质相互作用方面很有效，但不足以鉴定动态相互作用蛋白。在本章中，我们描述了通过将新的样品制备方法与基于SILAC的定量分析相结合来鉴定蛋白质复合物动态相互作用蛋白的综合策略。
