NEW PROTEOMIC REAGENT SYNTHESIS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 细胞机制分析中心建立了一个新的蛋白质组学试剂合成核心。在开发用于检测生物刺激后蛋白质水平变化的新型Zdye多重荧光蛋白染料的过程中，Grieco教授在荧光染料合成和制备针对各种官能团的染料变体方面积累了相当丰富的专业知识。 Grieco教授将指导该核心的各个方面，该核心将提供基于新Zdye荧光团家族的新的荧光蛋白质组试剂。 本核心描述中介绍了新的差异活性蛋白质谱分析（dABPP）试剂，本核心生产的差异巯基氧化还原蛋白质分析（dThiRPa）试剂将在Singel COBRE项目（项目-3）中进行表征。 项目1、2和4将大量使用这些新的dABPP和dThiRPA试剂。 ABPP领域正在迅速扩大，以确定新的反应基团，用于确定新类别酶活性的变化。 将开发用于所有酶活性类别的三功能捕获试剂，这些酶活性类别将用于捕获标记的蛋白质，以鉴定其活性位点区域。 这些独特的差异试剂将提供给中心的所有用户，无论他们是否有COBRE项目。 此外，还需要Z染料的附加颜色，以便可以同时进行更多变量的微分分析。 因此，我们预计这个核心在整个COBRE项目期间将保持忙碌，在后期制造尚未设计的试剂。 基本技术需要用具有适当反应性的不同颜色的荧光染料以各种方式处理的细胞中的蛋白质组分进行共价标记，混合标记的蛋白质并将其在2D凝胶上分离，如提案主体所述。激光扫描揭示了哪些蛋白质由于每个样品的不同生物处理而发生了变化。与其他蛋白质组学技术相比，2D凝胶方法具有显著的优势，即大多数蛋白质翻译后修饰将修饰形式转移到凝胶上的新位置（Halligan. Ruotti等人2004; Kumar. Khachane等人，2004年）。从差异分析的角度来看，这是最好的情况，因为新斑点的出现相对容易检测。 蛋白质翻译后修饰控制了很大一部分的生物学机制，然而，它们的检测已被证明是有问题的替代蛋白质组学方法，特别是当监测蛋白质翻译后修饰的全局变化。本COBRE提案中概述的dABPP和dthiRP方法利用2D凝胶方法的优势，提供每种蛋白质形式的相对量的全局图，并允许捕获感兴趣的蛋白质，以在质谱仪中进行鉴定，并通过检测标记位点进行活性位点鉴定。 2D凝胶技术存在许多公认的局限性，密歇根州立大学一直在努力将最佳可用技术联合收割机结合起来，并进行改进，以克服2D凝胶技术在全球差异蛋白质组学分析中的过去局限性。竞争的商业GE DIGE染料先前已经通过克服凝胶-凝胶再现性的问题提供了重大改进，但是具有有限的灵敏度，并且用DIGE染料标记可以降低凝胶图案上标记蛋白质的回收率。新的Z染料被设计成高度水溶性的，以提高标记蛋白质在其等电点的溶解度，从而可以加载更多的蛋白质用于更灵敏的分析，并且高的Z染料水溶性倾向于提高标记蛋白质的回收率并在2DE中从第一维转移到第二维。

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.