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

尽管2000年首次报道了植物的基因组序列，但在随后的几年里，我们还没有能够完全理解这些基因如何共同作用，使植物生长、发育和抵御干旱等环境压力。植物（与动物不同）不能通过移动来避免它们在环境中面临的挑战，我们需要新的知识来调节植物生长并维持作物产量的提高。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发育的分子机制；这些信息将帮助我们找到维持作物产量的方法，这对我们的食物、燃料、住所和衣物至关重要。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，其基本目标是消除质谱仪的神秘性，并帮助公众了解这些仪器如何提供惊人的灵敏度和能力，以检测我们环境中少量的有益和有害化合物。一门名为“全球变暖、篝火和蛋白质：它们有什么共同之处？”的新高中课程已经被开发出来，它的练习邀请学生体验科学家在探索植物的遗传学和生物化学时探索未知的方式，并在全球范围内绘制出分子、植物和人类的相互作用。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一个，这表明磷蛋白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由新发现的植物肽激素/受体激酶同源对引发的快速蛋白质磷酸化变化的体内功能，该对调节质子挤压和细胞扩增的速率。稳定同位素辅助非靶向“发现”测量使用高分辨率轨道阱串联质谱仪进行，然后使用化学合成的重同位素标记磷酸肽标准物和三重四极杆串联质谱仪进行靶向测量。利用含有磷酸化氨基酸突变的拟南芥植物进行反向遗传实验，为特定磷酸位点的植物功能提供了最终测试。计算方法将用于整理在大量环境和遗传扰动下执行的数据。这些基于网络的聚类大数据集的计算方法旨在获得基于蛋白激酶的信号通路的更全面的图像。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。

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.