EAGER: A General Method to Map Protein Phosphorylation Networks

这个急切的项目和即将开发的方法将多肽质谱学的最新进展与定量遗传学相结合，作为一种适用于任何生物体和任何翻译后修饰的高通量、低成本的分析方法，对更广泛的科学界具有潜在的价值。该项目将为研究生提供蛋白质组学、数量遗传学以及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利用翻译后机制，这种机制可以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。已知的修饰有很多种；研究最多的是磷酸化。植物逆境和发育的许多方面都受蛋白质磷酸化的调控。最近的蛋白质组学研究表明，磷酸化是动态的，广泛存在于整个蛋白质组中。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个蛋白激酶和磷酸酶的酶-底物关系图。这个急切的项目将开发出一种方法来迎接这一重大挑战。具体目标包括：1)建立大约50种已知经历生物胁迫诱导的特定位置的磷酸化变化的蛋白质的分析；2)定位调节每个位置的磷酸化水平的数量性状基因(QTL)；以及3)验证15个QTL下的选定基因的功能。所有数据(例如，原始MS谱、m/z值、离子强度、同位素比和层析保留时间)将通过项目网站和根据请求提供。

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.