EAGER: A General Method to Map Protein Phosphorylation Networks

该EAGER项目和将开发的方法结合了肽质谱与定量遗传学的最新进展，作为一种适用于任何生物体和任何翻译后修饰的高通量、低成本检测方法，对更广泛的科学界具有潜在价值。该项目将为一名研究生提供蛋白质组学、数量遗传学、植物生理学和分子生物学方面的培训。植物做出的大多数决定都是利用翻译后机制来感知环境或植物其他细胞的变化，并导致转录变化。一种主要的翻译后机制是蛋白质的可逆翻译后修饰。有许多已知的修改；研究得最好的是磷酸化。植物胁迫和发育的许多方面都受蛋白磷酸化的调控。最近的蛋白质组学研究表明，磷酸化是动态的，并且在整个蛋白质组中广泛存在。蛋白质磷酸化研究的一个重大技术挑战是开发一种方法来绘制每种蛋白激酶和磷酸酶的酶-底物关系。这个EAGER项目将开发一种方法来应对这一巨大挑战。具体目标包括：1)开发约50种已知在特定位点经历生物应激诱导的磷酸化变化的蛋白质的检测方法；2)定位调控每个位点磷酸化水平的数量性状位点（QTL）；3)验证15个QTL所选基因的功能。所有数据（例如，原始质谱，m/z值，离子强度，同位素比率和色谱保留时间）将通过项目网站提供，并应要求提供。

This EAGER project and the methods that will be developed combine recent advances in peptide mass spectrometry with quantitative genetics and has potential value to the broader scientific community as a high-throughput, low-cost assay that is applicable to any organism and any post-translational modification. The project will provide training for a graduate student in proteomics, quantitative genetics and plant physiology and molecular biology.Most decisions made by plants utilize post-translational mechanisms that sense changes in the environment or in other cells of the plant, and that lead to transcriptional changes. A major kind of post-translational mechanism is the reversible post-translational modification of proteins. There are many known modifications; the best studied is phosphorylation. Many aspects of plant stress and plant development are regulated by protein phosphorylation. Recent proteomic surveys have shown that phosphorylation is dynamic and widespread throughout the proteome. A technical grand challenge for research on protein phosphorylation is to develop a method for mapping the enzyme-substrate relationships for every protein kinase and phosphatase. This EAGER project will develop a method to meet this grand challenge. Specific objectives include 1) developing assays for approximately 50 proteins known to undergo biotic stress-induced changes in phosphorylation at specific sites; 2) mapping quantitative trait loci (QTL) that regulate the levels of phosphorylation at each site; and, 3) validating the function of selected genes underlying 15 QTL. All data (e.g., raw MS spectra, m/z values, ion intensities, isotope ratios, and chromatography retention times) will be available through the project website and upon request.