NEW PROTEOMIC REAGENT SYNTHESIS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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Zdy多重荧光蛋白染料的过程中，Grieco教授在荧光染料的合成和针对各种官能团的染料变体的制备方面积累了丰富的专业知识。格里科教授将指导这一核心的所有方面，该核心将提供基于新的ZdyFluobore家族的新的多色荧光蛋白质组试剂。本核心描述介绍了新的基于差异活性的蛋白质分析试剂(DABPP)，并将在Singel Cobre项目(Project-3)中对该核心公司生产的差异硫醇氧化还原蛋白分析(DThiRPA)试剂进行表征。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，以确定新的酶活性类别的变化。将为所有酶活性类别开发三功能捕获试剂，这些试剂将用于捕获标记蛋白质以确定其活性部位区域。这些独特的差示试剂将向该中心的所有用户提供，无论他们是否有Cobre项目。此外，还需要增加Zdyes的颜色，以便可以同时进行更多变量的差示分析。因此，我们预计该核心将在整个Cobre项目期间保持忙碌状态，在后期生产尚未设计的试剂。 基本技术需要用具有合适反应性的不同颜色的荧光染料以共价方式标记细胞中的蛋白质组分，混合标记的蛋白质并将其在2D凝胶上分离，如提案正文所述。激光扫描揭示了哪些蛋白质由于每个样本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等人的研究成果。2004年；库马尔。Khachane等人。2004年)。从差异分析的角度来看，这是最好的案例情景，因为新斑点的出现相对容易被检测到。 蛋白质翻译后修饰控制着很大一部分生物学机制，然而它们的检测已经被证明是替代蛋白质组学方法的问题，特别是在监测蛋白质翻译后修饰的全球变化时。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数量的全球图谱，并将允许捕获感兴趣的蛋白质，以便在质谱仪中进行鉴定，并通过检测标记的位点来鉴定活性部位。 2D Gel技术有许多公认的局限性，密歇根州立大学已经努力结合最好的可用技术，并进行改进，以克服2D Gel技术在全球差异蛋白质组分析中的过去限制。与之竞争的商用GE dige染料以前通过克服凝胶-凝胶重现性问题提供了重大改进，但灵敏度有限，用dige染料标记可能会降低凝胶图案上标记的蛋白质的回收率。新的Zdyes设计为高度水溶性，以提高标记蛋白质在其等电点的溶解度，从而可以负载更多的蛋白质进行更灵敏的分析，并且Zdyy的高水溶性倾向于提高标记蛋白质的回收率，并在2DE中从一维转移到第二维。

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.