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

虽然植物的基因组序列在2000年首次被报道，但在接下来的几年里，我们还不能完全理解这些基因是如何共同作用的，使植物能够生长、发育和抵御干旱等环境压力。植物(与动物不同)不能移动来避免它们在环境中面临的挑战，需要新的知识来使我们能够调节植物的生长并维持作物产量的提高。这个项目将有助于阐明一组重要的植物蛋白质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方法来维持对我们的食物、燃料、住所和衣服至关重要的作物产量。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，基本目标是解开质谱仪的神秘面纱，帮助公众了解这些仪器如何能够提供惊人的灵敏度和力量来检测我们环境中的少量好的和坏的化合物。一项新的高中课程名为“全球变暖、篝火和蛋白质：它们有什么共同之处？”已经开发了，其练习邀请学生体验科学家探索未知事物的方式，因为他们探索植物的遗传学和生物化学，并绘制出分子、植物和人类在全球层面上的相互作用。在拟南芥和其他植物的基因组中，蛋白激酶是所有基因家族中最大的，这表明磷蛋白质组的复杂性仍然知之甚少。这个项目的目标是通过开发和应用基于质谱学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转导的理解。生物学的重点是量化和评估由新发现的植物多肽激素/受体激酶同源对引发的蛋白质快速磷酸化变化在体内的功能，该对调控质子挤出和细胞扩张的速度。使用基于Orbitrap的高分辨率串联质谱仪进行稳定同位素辅助的非定向“发现”测量，然后使用化学合成的重同位素标记的磷肽标准和三重四极串联质谱仪进行定向测量。利用含有磷酸化氨基酸突变的拟南芥植物进行的反向遗传实验为特定的亚磷酸盐提供了植物内功能的最终测试。将使用一种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面的图景。总体而言，这些实验将确定关键的生长调节信号通路网络，以及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.