Global Measurement of Turnover of Plant Proteins

作者：加里·L·尼尔斯图恩(明尼苏达大学)和大卫·安古洛(德保罗大学)科学目标和方法：这项研究的目标是建立稳定的植物蛋白质同位素标记方法，以确定在不断变化的发育和环境条件下各种不同蛋白质的绝对周转率(半衰期)，并研究特定突变对蛋白质周转率的影响。几种不同的标记植物蛋白质的方法，包括在2H2O、13CO2或15N-硝酸铵存在下生长植物的一般方法，以及使用特定的稳定同位素标记的氨基酸、糖或其他氨基酸前体标记植物蛋白质的方法。特定蛋白质在预标记期后稳定同位素的损失将被用来确定每种方法对进一步研究的适用性。由于标记方法和程序引起的正常植物功能的变化将通过微阵列分析对基因表达变化的测量来监测。已知的以不同速度降解的蛋白质的周转率以及在蛋白质降解途径中改变的突变体的使用将用于测试所开发程序的适用性。预期更广泛的影响：该项目将支持和培训博士后科学家和研究生，他们将接触到功能蛋白质组学的独特多学科方法。将通过研究生的互访和互访来鼓励国际活动。将在第二年建立一个培训方案，以便在制定新方法时传授知识，特别是有针对性地努力接触到那些传统上为少数群体服务的机构和/或那些少数群体入学率较高的机构。最后，该项目将致力于开发一种全球蛋白质图谱的独特方法，并产生将被广泛应用并作为植物研究界的一项使能技术的资源。可在待开发的项目网站上访问数据，并将在TAIR存放和访问方法和方案。

PI: Jerry D. Cohen (University of Minnesota) CoPI: William M. Gray (University of Minnesota) Collaborators: Gary L. Nelsestuen (University of Minnesota) and David Angulo (DePaul University)Scientific objectives and approaches: The objectives of the research are to develop robust methods for stable isotope labeling of plant proteins to determine absolute rates of turnover (the half-life) for a wide number of different proteins under changing developmental and environmental conditions and to study the effects of specific mutations on rates of protein turnover. Several different methods for labeling plant proteins, including general methods based on growing plants on 2H2O, in the presence of 13CO2, or on 15N-ammonium nitrate, as well as methods where a specific stable isotope labeled amino acid, sugar or other amino acid precursor will be used to label plant proteins. Loss of stable isotope from specific proteins after a pre-labeling period will be used to determine the suitability of each method for further studies. Changes to normal plant function due to the labeling method and procedures will be monitored by measurement of gene expression changes by microarray analyses. The turnover for proteins known to be degraded at different rates and use of mutants altered in their pathways for protein degradation will be used to test the suitability of the procedures developed. Expected broader impacts: This project will support and train postdoctoral scientists and graduate students who will be exposed to a unique multidisciplinary approach to functional proteomics. International activities will be encouraged through exchange visits by the graduate students and by hosting reciprocal visits. A training program will be established in the second year to transfer knowledge of new methods as they are developed, with a particular targeted effort to reach institutions that are either traditionally minority-serving and/or those with significant minority enrollment. Finally, the project will work towards developing a unique approach to global protein profiling and generating the resources that will be widely applicable and serve as an enabling technology to the plant research community. Data can be accessed at the project website to be developed and methods and protocols will be deposited and accessed at TAIR.