Comparative Proteome Analysis of Protein Expression and Phosphorylation in Arabidopsis

精确控制调节蛋白和效应蛋白的表达水平和磷酸化状态对许多生物过程至关重要，如细胞周期、昼夜节律和激素反应。迄今为止，蛋白质组范围内的蛋白质表达和磷酸化分析技术尚未在植物中建立。本项目旨在通过系统研究模式植物拟南芥中受植物激素脱落酸调控的蛋白表达水平或磷酸化状态，开发、应用和建立新的比较蛋白质组学工具。同位素编码亲和标记和代谢标记将用于蛋白质表达的定量。磷酸化状态的蛋白质将通过富集磷酸化肽后的质谱测定。通过分析蛋白质的表达和磷酸化状态，可以识别出磷酸化发生变化而表达没有相应变化的蛋白质，反之亦然。该项目最有可能导致发现新的脱落酸调节蛋白，并发展关于这些蛋白在脱落酸信号传导中的可测试假设。更广泛的影响：该项目开发的技术将成为植物生物学家系统分析蛋白质表达和磷酸化的有用工具。本项目确定的所有体内蛋白磷酸化位点将发布在PlantsP数据库（http://plantsp.sdsc.edu）上。该项目制定的详细协议将发布在一个可公开访问的网站上。该项目将为EPSCoR州的本科生、研究生和博士后提供极好的机会，以进行定量蛋白质组学的最新研究。

Precise control of the expression level and phosphorylation state of regulator and effector proteins is critical for many biological processes such as the cell cycle, circadian rhythm, and hormone responses. To date, technologies for proteome-wide analysis of protein expression and phosphorylation have not been established in plants. The objective of this project is to develop, apply, and establish new tools for comparative proteomics by systematically studying proteins whose expression level or phosphorylation state or both are regulated by the plant hormone abscisic acid in the model plant Arabidopsis. Isotope-coded affinity tagging and metabolic labeling will be applied for quantitation of protein expression. Phosphorylation state of proteins will be determined by mass spectrometry after enrichment of phosphopeptides. By analyzing both protein expression and phosphorylation state, proteins that undergo a change in phosphorylation without a corresponding change in expression or vice versa can be identified. This project is most likely to lead to discovery of novel abscisic acid-regulated proteins and the development of testable hypotheses about these proteins in abscisic acid signaling. Broader Impacts: The technologies developed from this project will serve as useful tools for plant biologists to systematically analyze protein expression and phosphorylation in their favorite systems. All in vivo protein phosphorylation sites determined in this project will be posted on the PlantsP database (http://plantsp.sdsc.edu). Detailed protocols developed from this project will be posted on a publicly accessable website. This project will provide excellent opportunities to train undergraduates, graduate students, and postdoctoral fellows from an EPSCoR state to perform state-of-the art research in quantitative proteomics.