Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota.
College of Biological Sciences Center for Mass Spectrometry and Proteomics, University of Minnesota, Minneapolis, Minnesota.
Curr Protoc Immunol. 2020 Sep;130(1):e104. doi: 10.1002/cpim.104.
In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein-protein interactions, and changes in post-translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease. © 2020 The Authors. Basic Protocol 1: Quantifying protein phosphorylation via immunoblotting and near-infrared imaging Alternate Protocol: Visualizing immunoblots using chemiluminescence Basic Protocol 2: Enriching target proteins and isolation of protein complexes by immunoprecipitation Support Protocol: Covalent conjugation of antibodies to functionalized beads Basic Protocol 3: Quantifying proteins and post-translational modifications by targeted mass spectrometry.
在本文中，我们描述了使用药理学和遗传学工具，结合免疫印迹（Western blot）和靶向质谱，来定量分析酪氨酸激酶介导的免疫信号和细胞激活。将 ATP γ 磷酸转移到蛋白质酪氨酸残基上，激活了调节所有细胞分化、存活和效应功能的信号级联反应，在免疫抗原受体、极化和其他信号通路中具有独特的作用。确定酪氨酸激酶的底物和支架相互作用对于揭示和治疗性操纵免疫调节机制至关重要。这些效应的幅度和动力学的定量分析在实验上越来越容易实现，并且对于预测治疗药物的复杂下游效应以及构建计算模型变得越来越重要。其次，定量分析越来越受到审稿人和期刊编辑的期望，并且对生物学重复的统计分析可以增强实验严谨性和可重复性的主张。在这里，我们概述了在细胞中扰动酪氨酸激酶活性和定量测定裂解物和免疫沉淀物中蛋白质磷酸化的方法。免疫印迹技术是探测蛋白质丰度、蛋白质-蛋白质相互作用和翻译后修饰变化的动态的指南。免疫沉淀的蛋白质复合物也可以进行靶向质谱分析，以探测新的修饰位点以及无法通过免疫印迹解析的多修饰或研究较少的蛋白质。这些方案共同构成了一个框架，用于确定酪氨酸激酶对细胞激活的独特贡献，并阐明健康和疾病中免疫细胞调节的机制。© 2020 作者。基本方案 1：通过免疫印迹和近红外成像定量测定蛋白质磷酸化替代方案：使用化学发光可视化免疫印迹基本方案 2：通过免疫沉淀富集靶蛋白和分离蛋白质复合物支持方案：抗体与功能化珠共价偶联基本方案 3：通过靶向质谱定量测定蛋白质和翻译后修饰
