Endler Anne, Baginsky Sacha
Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Saale, Germany.
Methods Mol Biol. 2011;775:283-96. doi: 10.1007/978-1-61779-237-3_15.
The posttranslational modification of proteins is important for the regulation of enzymatic activity, protein half-life, and interaction with other molecules. One of the best understood posttranslational modifications is the reversible phosphorylation of proteins at serine, threonine, or tyrosine residues. These phosphoamino acids are relatively stable in acidic solutions, and their comprehensive identification by mass spectrometry is, therefore, feasible. Phosphoproteomics-type experiments require some modifications in the sample preparation, mass spectrometry setup, and software-based data interpretation compared to standard proteomics workflows. Furthermore, phosphoproteome analyses are incompatible with long organelle isolation procedures prior to analysis, because of the highly dynamic nature of regulatory phosphorylations. In this chapter, we provide a detailed step-by-step overview of the complex experimental setup required for successful chloroplast phosphoproteome analysis, report our experience with existing methods, and comment on their application in the field.
蛋白质的翻译后修饰对于酶活性调节、蛋白质半衰期以及与其他分子的相互作用至关重要。目前了解最为透彻的翻译后修饰之一是蛋白质在丝氨酸、苏氨酸或酪氨酸残基上的可逆磷酸化。这些磷酸化氨基酸在酸性溶液中相对稳定，因此通过质谱对其进行全面鉴定是可行的。与标准蛋白质组学工作流程相比，磷酸化蛋白质组学类型的实验在样品制备、质谱设置以及基于软件的数据解读方面需要一些改进。此外，由于调节性磷酸化具有高度动态性，磷酸化蛋白质组分析与分析前长时间的细胞器分离程序不兼容。在本章中，我们详细逐步概述了成功进行叶绿体磷酸化蛋白质组分析所需的复杂实验设置，报告了我们对现有方法的经验，并对其在该领域的应用进行了评论。
