Eppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA.
Mass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, NE, USA.
Methods Mol Biol. 2023;2701:185-197. doi: 10.1007/978-1-0716-3373-1_12.
Immunoprecipitation-mass spectrometry (IP-MS) is a versatile tool to probe for global protein-protein interactions (PPIs) in biological samples. Such interactions coordinate complex biological processes, such as the DNA damage response (DDR). Induction of DNA damage activates signaling networks where posttranslational modifications cause PPI that facilitate DNA repair and cell cycle coordination. Protein interactome profiling of DDR sensors, transducers, and effectors has the potential to identify novel DDR mechanisms that could advance our understanding and treatment of diseases associated with DDR defects, such as cancer. The protocol described here is a routine PPI analysis procedure that can be performed on samples stimulated with DNA damage. All processes and reagents are optimized for maximum sensitivity on the interactome and minimal contamination for the mass spectrometer.
免疫沉淀-质谱（IP-MS）是一种通用的工具，可用于探测生物样本中的全局蛋白质-蛋白质相互作用（PPIs）。这种相互作用协调着复杂的生物过程，如 DNA 损伤反应（DDR）。DNA 损伤的诱导激活了信号网络，其中翻译后修饰导致 PPI，从而促进 DNA 修复和细胞周期协调。DDR 传感器、转导器和效应器的蛋白质相互作用组谱分析有可能识别新的 DDR 机制，从而推进我们对与 DDR 缺陷相关疾病（如癌症）的理解和治疗。这里描述的方案是一种可在受 DNA 损伤刺激的样品上进行的常规 PPI 分析程序。所有过程和试剂都经过优化，以最大限度地提高互作组的灵敏度和最小化质谱仪的污染。
