NEW PROTEOMIC REAGENT SYNTHESIS

新的蛋白质组试剂合成

基本信息

批准号：
8167557
负责人：
PAUL A GRIECO
金额：
$ 34.26万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8167557
关键词：
Active Sites Appearance Biological Cell Fraction Centers of Research Excellence Color Computer Retrieval of Information on Scientific Projects Database Detection Dimensions Dyes Family Fluorescent Dyes Funding Gel Global Change Grant Institution Isoelectric Point Label Lasers Maps Methodology Methods Monitor Oxidation-Reduction Pattern Positioning Attribute Post-Translational Protein Processing Process Protein Analysis Proteins Proteomics Reagent Recovery Relative (related person)Reproducibility Research Research Personnel Resources Sampling Scanning Site Solubility Source Spottings Stimulus Sulfhydryl Compounds Technology United States National Institutes of Health Variant Water activity-based protein profiling aerobic respiration control protein base bropirimine design enzyme activity functional group interest mass spectrometer professor water solubility

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。细胞机制分析中心建立了新型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 荧光团家族的新多色荧光蛋白质组试剂。本核心描述中介绍了新的基于活性的差异蛋白质分析 (dABPP) 试剂，并且该核心还生产的差异硫醇氧化还原蛋白质分析 (dThiRPa) 试剂将在 Singel COBRE 项目 (Project-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 领域正在迅速扩展，以识别新的反应基团，从而识别新类别酶活性的变化。将为所有酶活性类别开发三功能捕获试剂，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Zdyes的附加颜色，以便可以同时进行更多变量的差异分析。因此，我们预计该核心将在整个 COBRE 项目期间保持忙碌，并在后期制造尚未设计的试剂。基本技术需要共价标记来自用具有适当反应性的不同颜色荧光染料以各种方式处理的细胞的蛋白质级分，混合标记的蛋白质并在 2D 凝胶上分离它们，如提案正文中所述。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 凝胶方法具有显着的优势，即大多数蛋白质翻译后修饰会将修饰形式转移到凝胶上的新位置（Halligan. Ruotti 等人，2004 年；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到。蛋白质翻译后修饰控制着很大一部分生物机制，但事实证明，它们的检测对于替代蛋白质组学方法来说是有问题的，特别是在监测蛋白质翻译后修饰的整体变化时。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相对量的全局图，并允许捕获感兴趣的蛋白质，以便在质谱仪中进行鉴定，并活性-通过检测标记位点进行位点识别。 2D 凝胶技术存在许多公认的局限性，密歇根州立大学已努力结合现有的最佳技术并进行改进，以克服 2D 凝胶技术过去在全球差异蛋白质组分析中的局限性。竞争性商业 GE DIGE 染料之前通过克服凝胶-凝胶重现性问题提供了重大改进，但灵敏度有限，并且用 DIGE 染料进行标记会降低凝胶图案上标记蛋白质的回收率。新的 Zdye 被设计为高度水溶性，以增强标记蛋白质在等电点的溶解度，从而可以加载更多蛋白质以进行更灵敏的分析，并且 Zdye 的高水溶性往往会提高标记蛋白质的回收率和从2DE 中的第一维到第二维。