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细胞周期、昼夜节律和激素反应等许多生物学过程至关重要。到目前为止，在植物中还没有建立蛋白质组范围的蛋白质表达和磷酸化分析技术。本项目的目的是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质的表达水平和/或磷酸化状态，来开发、应用和建立新的比较蛋白质组学工具。同位素编码的亲和标记和代谢标记将用于蛋白质表达的定量。蛋白质的磷酸化状态将通过浓缩后的磷酸多肽通过质谱分析来确定。通过分析蛋白质表达和磷酸化状态，可以识别经历了磷酸化变化而没有相应表达变化的蛋白质，或者相反的蛋白质。这个项目最有可能导致发现新的脱落酸调节蛋白，并开发关于这些蛋白在脱落酸信号转导中的可测试假说。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他们最喜欢的系统中的蛋白质表达和磷酸化的有用工具。该项目中确定的所有体内蛋白质磷酸化位点将公布在植物P数据库(http://plantsp.sdsc.edu).)上从这个项目制定的详细协议将张贴在一个可公开访问的网站上。这个项目将提供极好的机会来培训EPSCoR州的本科生、研究生和博士后研究员进行最先进的定量蛋白质组学研究。

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.