Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, United States.
Department of Biology, Genetics Institute, University of Florida, Gainesville, FL, United States.
Methods Enzymol. 2022;676:347-368. doi: 10.1016/bs.mie.2022.06.004. Epub 2022 Jul 29.
Among all post-translational modifications of proteins, phosphorylation is one of the most common and most studied. Since plants are sessile organisms, many physiological processes on which their survival depends are regulated by phosphorylation and dephosphorylation. Understanding the extent to which a plant proteome is phosphorylated at specific developmental stages and/or under certain environmental conditions is essential for identifying molecular switches that regulate physiological processes and responses. While most phosphoproteomic workflows proposed in the literature provide tools to exclusively analyze phosphorylated proteins, it is imperative to examine both the proteome and the phosphoproteome to reveal the true complexity of a biological process. Here we describe a mass spectrometry-based phosphoproteomics workflow to analyze both total and phosphorylated proteins. Our method includes phenol-based protein extraction, as well as techniques to measure the quantity and quality of protein extracts. In addition, we compare in detail the efficiency and suitability of in-gel and in-solution trypsin digestion methods. A metal oxide affinity chromatography technique for rapid and efficient enrichment of phosphorylated peptides and an LC-MS/MS method for analysis of the phosphorylated peptides are described. Finally, we present and discuss the results generated by applying this workflow to our study of the C to CAM transition in the common ice plant (Mesembryanthemum crystallinum). Overall, our workflow provides robust methods for the identification of phosphoproteins and total proteins. It can be broadly applied to many other organisms and sample types, and the results provide a more accurate picture of the molecular switches that regulate different biological processes.
在蛋白质的所有翻译后修饰中，磷酸化是最常见和研究最多的修饰之一。由于植物是固着生物，许多依赖于其生存的生理过程受到磷酸化和去磷酸化的调节。了解植物蛋白质组在特定发育阶段和/或特定环境条件下被磷酸化的程度，对于识别调节生理过程和反应的分子开关至关重要。尽管文献中提出的大多数磷酸蛋白质组学工作流程提供了专门分析磷酸化蛋白质的工具，但检查蛋白质组和磷酸蛋白质组对于揭示生物学过程的真正复杂性是必不可少的。在这里，我们描述了一种基于质谱的磷酸蛋白质组学工作流程，用于分析总蛋白和磷酸化蛋白。我们的方法包括基于酚的蛋白质提取，以及用于测量蛋白质提取物的数量和质量的技术。此外，我们详细比较了凝胶内和溶液中胰蛋白酶消化方法的效率和适用性。描述了一种用于快速高效富集磷酸肽的金属氧化物亲和层析技术以及用于分析磷酸肽的 LC-MS/MS 方法。最后，我们介绍并讨论了将此工作流程应用于我们对常见冰叶日中花（Mesembryanthemum crystallinum）从 C 到 CAM 转变的研究中生成的结果。总的来说，我们的工作流程为鉴定磷酸化蛋白和总蛋白提供了可靠的方法。它可以广泛应用于许多其他生物体和样本类型，并且结果提供了更准确的调节不同生物学过程的分子开关的图片。
