NEW PROTEOMIC REAGENT SYNTHESIS

新的蛋白质组试剂合成

• 批准号: 8359567
• 负责人: PAUL A GRIECO
• 金额: $ 33.91万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8359567
• 关键词: Active Sites; Appearance; Biological; Cell Fraction; Centers of Research Excellence; Color; Detection; Dimensions; Dyes; Family; Fluorescent Dyes; Funding; Gel; Global Change; Grant; Isoelectric Point; Label; Lasers; Maps; Methodology; Methods; Monitor; National Center for Research Resources; Oxidation-Reduction; Pattern; Positioning Attribute; Post-Translational Protein Processing; Principal Investigator; Process; Protein Analysis; Proteins; Proteomics; Reagent; Recovery; Relative (related person); Reproducibility; Research; Research Infrastructure; Resources; Sampling; Scanning; Site; Solubility; Source; Spottings; Stimulus; Sulfhydryl Compounds; Systems Biology; Technology; United States National Institutes of Health; Variant; Water; activity-based protein profiling; aerobic respiration control protein; base; bropirimine; cost; design; enzyme activity; functional group; interest; mass spectrometer; professor; water solubility

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Center for the Analysis of Cellular Mechanisms has establish a New Proteomics Reagent Synthesis Core. In the process of developing the new Zdye multiplex fluorescent protein dyes for detecting changes in protein levels after biological stimuli, Prof. Grieco has developed considerable expertise in fluorescent dye synthesis and preparing variations of the dyes targeted for various functional groups. Professor Grieco will direct all aspects of this Core, which will provide new multicolor fluorescent proteomic reagents based on the new Zdye fluorphore family. New differential Activity-Based Protein Profiling (dABPP) reagents are presented in this Core Description and differential Thiol Redox Protein Analysis (dThiRPa) reagents also produced by this Core will be characterized in the Singel COBRE Project (Project-3). Projects 1, 2 and 4 will make substantial use of these new dABPP and dThiRPA reagents. The ABPP field is expanding rapidly to identify new reactive groups for identifying changes in new classes of enzyme activities. Trifunctional capture reagents will be developed for all of the enzyme activity classes that will be used to capture the labeled proteins for identification of their active site regions. These unique differential reagents will be made available to all users of the Center whether they have COBRE projects or not. In addition, there is a need for additional colors of Zdyes so that differential analysis of more variables can be carried out simultaneously. Thus, we expect that this Core will remain busy throughout the entire COBRE project period, in the later periods making reagents that have not been designed as yet. The basic technology entails covalently labeling protein fractions from cells treated in various ways with different-colored fluorescent dyes with suitable reactivity, mixing the labeled proteins and separating them on 2D gels, as described in the main body of the proposal. Laser scanning reveals which proteins have changed due to the different biological treatments of each sample. The 2D gel approach has the significant advantage, compared to other proteomics technologies, that most protein post-translational modifications shift the modified form to a new position on the gel (Halligan. Ruotti et al. 2004; Kumar. Khachane et al. 2004). From the perspective of differential analysis, this is a best case scenario because the appearance of a new spot is relatively easy to detect. Protein post-translational modifications control a very large fraction of biological mechanisms, however their detection has proven to be problematic for the alternative proteomics methodologies, particularly when monitoring global changes in protein post-translational modifications. The dABPP and dthiRP approaches outlined in this COBRE proposal take advantage of the strength of 2D gel methods to provide a global map of the relative amounts of each protein form and will allow the capture of the proteins of interest for identification in the mass spectrometer and active-site identification by detection of the labeled sites. There have been many well-recognized limitations to 2D gel technology and efforts have been mounted at MSU to combine the best available technology and to add improvements to overcome the past limitations in 2D gel technology for global differential proteomic analysis. Competing commercial GE DIGE dyes have previously provided a major improvement by overcoming problems of gel-gel reproducibility, but have limited sensitivity and labeling with DIGE dyes can reduce the recovery of labeled proteins on the gel patterns. The new Zdyes are designed to be highly water soluble to enhance the solubility of the labeled proteins at their isoelectric points, so that more protein can be loaded for more sensitive analysis and the high Zdye water solubility tends to enhance recovery of labeled proteins and transfer from the first to the second dimension in 2DE.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 细胞机制分析中心已建立了一种新的蛋白质组学试剂合成核心。在开发新的ZDYE多重荧光蛋白染料以检测生物刺激后蛋白质水平的变化的过程中，Grieco教授在荧光染料合成和制备针对各种功能组的染料的变化方面开发了相当大的专业知识。 Grieco教授将指导该核心的各个方面，该核心将根据新的Zdye Fluorphore家族提供新的多色荧光蛋白质组学试剂。 在此核心描述中介绍了新的基于差异活性的蛋白质分析（DABPP）试剂，该核心也将在Singel Cobre Project（Project-3）中表征该核心产生的差异硫醇氧化还原蛋白分析（DTHIRPA）试剂。 项目1、2和4将大量使用这些新的DABPP和DTHIRPA试剂。 ABPP领域正在迅速扩展，以识别新的反应组，以识别新的酶活性类别的变化。 将针对所有酶活性类别开发三功能捕获试剂，该试剂将用于捕获标记的蛋白质以识别其活性位点区域。 这些独特的差异试剂将用于中心的所有用户，无论它们是否有毛病项目。 另外，需要其他颜色的Zdyes颜色，因此可以同时对更多变量进行差分分析。 因此，我们预计，在整个毛线项目期间，在尚未设计的试剂中，这种核心将保持繁忙。 基本技术需要以各种方式与不同颜色的荧光染料处理的细胞共价标记蛋白质级数，并具有适当的反应性，将标记的蛋白质混合并在2D凝胶上分离，如提案的主体所述。激光扫描揭示了由于每个样品的不同生物学处理，哪些蛋白质发生了变化。与其他蛋白质组学技术相比，2D凝胶方法具有显着优势，即大多数蛋白质后翻译后修饰将修饰的形式转移到凝胶上的新位置（Halligan。Ruotti等，2004；Kumar。Khachane.Khachane等，2004）。从差分分析的角度来看，这是最好的情况，因为新斑点的外观相对容易检测到。 蛋白质后翻译修饰控制着很大一部分的生物学机制，但是事实证明，它们的检测对于替代蛋白质组学方法论是有问题的，尤其是在监测蛋白质后翻译后修饰的全球变化时。该毛刺提案中概述的DABPP和DTHIRP方法利用了2D凝胶方法的强度，提供了每种蛋白质形式的相对量的全球图，并将允许在质谱仪中捕获感兴趣的蛋白质以通过检测标记的位点识别质谱仪和主动Site识别。 2D凝胶技术已经有许多公认的局限性，并且已经在MSU上进行了努力，以结合最佳的技术并增加改进，以克服过去2D凝胶技术的过去局限性，以进行全球差异蛋白质组学分析。相互竞争的商业GE DIGE染料以前通过克服凝胶凝胶可重复性问题的问题提供了重大改进，但是敏感性有限，并且使用Dige染料标记可以减少凝胶模式上标记的蛋白质的回收率。新的ZDYES设计为高度水溶性，以增强标记的蛋白质在其等电点处的溶解度，因此可以加载更多的蛋白质以进行更敏感的分析，并且高ZDYE水溶性倾向于增强标记的蛋白质的恢复，并在2DE中从第一个尺寸转移到第二个尺寸。