An Isotope-Assisted Quantitative Phosphoproteomic Analysis of Signaling Pathways Initiated at the Plasma Membrane of Arabidopsis thaliana

同位素辅助对拟南芥质膜启动的信号通路进行定量磷酸化蛋白质组学分析

• 批准号: 1410164
• 负责人: Michael Sussman
• 金额: $ 134.73万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1410164&HistoricalAwards=false
• 关键词: Isotope; Assisted; Quantitative; Phosphoproteomic; Analysis

Although the genome sequence of a plant was first reported in 2000, in the ensuing years we have not yet been able to understand fully how these genes act together to allow plants to grow, develop and withstand environmental stresses like drought. Plants (unlike animals) cannot move to avoid the challenges they face in their environment, and new knowledge is needed to allow us to regulate plant growth and sustain improvements in crop yields. This project will help elucidate the molecular mechanisms by which a group of important plant proteins control plant cell growth and development; this information will help us develop methods to maintain the crop yields that are important for our food, fuel, shelter and clothing. This project will provide training for graduate students, and offer educational opportunities and activities for the community, with the basic goal of de-mystifying mass spectrometers and helping the public understand how these instruments can provide amazing sensitivity and power for detecting small amounts of good and bad compounds in our environment. A new high school curriculum called "Global Warming, Campfires and Proteins: What Do They Have in Common?" has been developed and its exercises invite students to experience the ways that scientists explore the unknown as they probe the genetics and biochemistry of plants, and map out the interplay of molecules, plants and humans at the global level. In the genome of Arabidopsis and other plants, protein kinases represent the largest of all gene families, pointing to a complexity in the phosphoproteome that remains poorly understood. The goal of this project is to increase our understanding of protein kinase mediated signaling by the development and application of mass spectrometric based isotope-assisted quantitative proteomic and computational tools. The biological emphasis is on quantifying and evaluating the in vivo function of rapid protein phosphorylation changes initiated by a newly discovered plant peptide hormone/receptor kinase cognate pair that regulates the rate of proton extrusion and cell expansion. Stable isotope-assisted untargeted "discovery" measurements are performed with a high resolution Orbitrap-based tandem mass spectrometer followed by targeted measurements using chemically synthesized heavy isotope labeled phosphopeptide standards and a triple quadrupole tandem mass spectrometer. Reverse genetic experiments using Arabidopsis plants containing mutations in the phosphorylated amino acids provide final tests of the in planta functions for specific phosphosites. A computational approach will be used to collate data performed under a large number of environmental and genetic perturbations. These network based computational methods of clustering large datasets are aimed at obtaining a more global picture of the protein kinase based signaling pathways. Overall, these experiments will identify networks of key growth regulating signaling pathways, as well as reveal important new insights on the molecular mechanism by which cells expand.

虽然植物的基因组序列在2000年首次被报道，但在随后的几年里，我们还无法完全理解这些基因是如何共同作用的，以使植物能够生长、发育和承受干旱等环境压力。 植物（与动物不同）无法移动以避免它们在环境中面临的挑战，需要新的知识来调节植物生长并持续提高作物产量。 该项目将有助于阐明一组重要植物蛋白控制植物细胞生长和发育的分子机制;这些信息将帮助我们开发维持作物产量的方法，这对我们的食物、燃料、住所和衣服很重要。该项目将为研究生提供培训，并为社区提供教育机会和活动，其基本目标是揭开质谱仪的神秘面纱，帮助公众了解这些仪器如何提供惊人的灵敏度和能力来检测我们环境中的少量好化合物和坏化合物。一个新的高中课程名为“全球变暖，篝火和蛋白质：他们有什么共同点？“已发展，其练习邀请学生体验科学家探索未知的方式，因为他们探索植物的遗传学和生物化学，并绘制出分子，植物和人类在全球一级的相互作用。 在拟南芥和其他植物的基因组中，蛋白激酶代表了所有基因家族中最大的家族，这表明磷酸化蛋白质组的复杂性仍然知之甚少。该项目的目标是通过开发和应用基于质谱的同位素辅助定量蛋白质组学和计算工具来增加我们对蛋白激酶介导的信号传导的理解。生物学的重点是量化和评估体内功能的快速蛋白磷酸化的变化，启动了一个新发现的植物肽激素/受体激酶同源对，调节质子挤出和细胞扩张的速度。稳定的同位素辅助的非靶向“发现”测量是用高分辨率的基于Orbitrap的串联质谱仪进行的，然后使用化学合成的重同位素标记的磷酸肽标准品和三重四极串联质谱仪进行靶向测量。使用含有磷酸化氨基酸突变的拟南芥植物的反向遗传实验提供了特定磷酸化位点的植物功能的最终测试。将使用计算方法来整理在大量环境和遗传扰动下进行的数据。这些基于网络的大型数据集聚类计算方法旨在获得基于蛋白激酶的信号通路的更全局的图像。总的来说，这些实验将确定关键生长调节信号通路的网络，并揭示细胞扩张的分子机制的重要新见解。