SGER: Proteomic Analysis of Multi-Gene Families

植物具有编码如此多的蛋白质的基因家族，以至于用于研究蛋白质水平表达的传统的基于抗体的方法变得几乎不可能。 由于关于发育阶段、亚细胞定位和环境条件的蛋白质含量的信息可以提供关于功能的重要线索，因此用于鉴定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学分析的工具。 这个项目是合理的支持，作为探索性研究的小额赠款，因为它涉及到未经测试的和新的想法的初步工作。程序开始与肽序列和差异标记的两套肽编码的质量标签。在混合两组后，采用免疫亲和色谱法分离携带表位的肽。这些通过串联质谱仪的反相色谱法进行解析，其中测定来自不同组的肽的相对峰强度并鉴定亲本蛋白。为了测试该方法，我们将从从表达克隆制备的已知肽混合物开始。将模拟混合物含有对抗体具有不同亲和力的肽或少数肽高度丰富的情况。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，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跨越植物物种。因此，这种新的方法可以用来发现作物中具有相同组织特异性和亚细胞定位的直向同源基因。那些直系同源物，也显示出平行的蛋白质水平的变化，相对于发育阶段和激素的线索和环境压力的反应可能共享类似的功能。因此，了解一个单独的蛋白质在一个良好的研究模式物种的功能，可以迅速倍增到了解其他植物物种的直系同源物的功能。相反，可以在模式物种中识别直系同源物进行更深入的研究，并将结果应用于其他植物物种，以提高作物产量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。它还将为主要研究者教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.