SGER: Proteomic Analysis of Multi-Gene Families

植物的基因家族编码如此之多的蛋白质，传统的基于抗体的蛋白质水平表达研究方法几乎是不可能的。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信息可以提供有关功能的重要线索，因此一种鉴定多基因家族蛋白质并测量其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是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学分析的工具。这个项目有理由获得探索性研究小额资助，因为它涉及未经测试和新颖的想法的初步工作。该程序从肽消化和两组肽的编码质量标签的差异标记开始。将两组混合后，采用免疫亲和层析分离含表位肽。这些都是通过反相色谱在线到串联质谱仪，从不同的集的肽的相对峰强度被确定和亲本蛋白鉴定。为了测试该方法，我们将从从表达克隆制备的已知多肽混合物开始。将模拟混合物中含有对抗体有不同亲和力的肽或其中一些非常丰富的情况。这项工作将使用拟南芥和水稻幼苗叶片制备的蛋白质提取物进行。虽然概念验证工作将集中在枯草杆菌蛋白酶基因家族上，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。多基因家族的同源性在植物物种中广泛存在。因此，这种新方法可用于揭示作物植物中具有相同组织特异性和亚细胞定位的同源基因。这些同源物也显示出与发育阶段和对激素信号和环境压力的反应有关的蛋白质水平的平行变化，可能具有相似的功能。因此，了解一个已被充分研究的模式物种中单个蛋白质的功能，可以迅速增加对其他植物物种中同源物功能的理解。相反，同源物可以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纤维和特种化学品的新来源。该项目将为研究生和本科生提供研究机会。它还将为首席研究员讲授的生物化学和蛋白质组学课程的讨论和解决问题提供数据。

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.