SGER: Proteomic Analysis of Multi-Gene Families

植物有编码如此多蛋白质的基因家族，以至于传统的基于抗体的方法研究蛋白质水平的表达几乎是不可能的。由于与发育阶段、亚细胞定位和环境条件有关的蛋白质含量的信息可以提供关于功能的重要线索，因此一种识别多基因家族的蛋白质并测量它们在植物组织中的相对水平的方法将是有价值的。在这个概念验证项目中开发的方法利用识别基因家族中共同表位的抗体作为分离家族成员进行蛋白质组分析的工具。这个项目作为探索性研究的小额赠款是合理的，因为它涉及到未经测试的新想法的前期工作。程序始于多肽消化和两组带有编码质量标签的多肽的差异标记。将两组表位混合后，用免疫亲和层析法分离抗原表位多肽。这些通过反相色谱在线串联质谱仪进行解析，在该质谱仪中，来自不同组的多肽的相对峰强度被确定，并且亲本蛋白质被鉴定。为了测试这种方法，我们将从已知的从表达克隆制备的多肽混合物开始。将模拟混合物含有与抗体具有不同亲和力的多肽或少数多肽高度丰富的情况。这项工作将使用从拟南芥和水稻幼苗叶片中提取的蛋白质进行。尽管概念验证工作将集中在枯草杆菌蛋白水解酶基因家族，但这种蛋白质组学方法将直接适用于许多其他多基因家族。多基因家族内的同源性在植物物种之间延伸。因此，这种新的方法可以用来发现具有相同组织特异性和亚细胞定位的作物中的同源基因。这些同源基因也显示出蛋白质水平在发育阶段以及对激素提示和环境压力的反应方面的平行变化，可能具有类似的功能。因此，对一个经过充分研究的模式物种中单个蛋白质功能的了解可以迅速扩大到了解直系物在其他植物物种中的功能。相反，可以确定一个直系同源基因，以便在模式物种中进行更深入的研究，并将结果应用于其他植物物种，以提高作物生产力或开发新的纤维和特种化学品来源。该项目将为研究生和本科生提供研究机会。它还将为首席研究人员教授的生物化学和蛋白质组学课程中的讨论和问题解决会议提供数据。

Plants have gene families encoding so many proteins that traditional antibody-based methods for studying protein level expression become nearly impossible. Since information about protein content with respect to developmental stage, subcellular localization and environmental conditions can give important clues regarding function, a method for identifying proteins of a multi-gene family and measuring their relative levels in plant tissues would be valuable. The approach to be developed in this proof of concept project makes use of antibodies that recognize common epitopes in a gene family as a tool to isolate family members for proteomic analysis. This project is justified for support as a Small Grant for Exploratory Research because it involves preliminary work on untested and novel ideas.The procedure starts with peptide digests and differential labeling of two sets of peptides with coded mass tags. After mixing the two sets, immunoaffinity chromatography is employed to isolate the epitope-bearing peptides. These are resolved by reverse phase chromatography in-line to a tandem mass spectrometer where relative peak intensities of the peptide from different sets are determined and parent proteins are identified. To test the method, we will start with known mixtures of peptides prepared from expressed clones. Situations in which mixtures contain peptides of varying affinity to antibody or where a few are highly abundant will be simulated. This work will be carried out using protein extracts prepared from Arabidopsis thaliana and rice seedling leaves.Although the proof of concept work will focus on the subtilisin protease gene family, this proteomics approach will be directly applicable to many other multi-gene families. Homology within multi-gene families extends across plant species. Therefore, this new approach can be used to uncover orthologous genes in crop plants that share the same tissue specificity and subcellular localization. Those orthologs that also show parallel changes in protein level with respect to developmental stage and responses to hormonal cues and environmental stresses are likely to share similar functions. Thus, understanding of the function of an individual protein in a well-studied model species can be multiplied rapidly toward understanding the function of orthologs in other plant species. Conversely, an ortholog can be identified for deeper study in a model species, and the results applied back to other plant species to increase crop productivity or to develop new sources of fiber and specialty chemicals. The project will provide research opportunities for graduate and undergraduate students. It will also provide data for discussions and problem-solving sessions in biochemistry and proteomics courses taught by the Principal Investigators.