Multiplex Fluorescent Dyes for Ultrasensitive Proteomics

描述（申请人提供）：该项目的广泛的长期目标是开发更强大的蛋白质组学检测技术，大大提高灵敏度，这将揭示生物刺激变化的全球模式（差异检测）蛋白质水平，翻译后修饰和酶活性的同时（使用6-12通道荧光染料（Zdye）检测），开发使用蛋白质同种型水平的模式来识别变化的生理状态的方法，并且更有效地靶向和获得可变蛋白质用于详细的质谱分析。本项目的具体目标和研究设计是：（1）扩大氨基活性Zdye和Z2染料的合成，这些染料在目前的第一阶段计划中已经开发并显示出可行性;（2）制备和测试与蛋白质硫醇反应以避免标记蛋白质沉淀的高度水溶性Zdye饱和标记物（这是用于2D凝胶检测的可用硫醇饱和标记的问题），以促进饱和标记的感兴趣点中每种类型的蛋白质的点拾取或靶向电洗脱，并提供额外的差异检测灵敏度，与Zdye氨基标记相比（与目前可用的氨基标记物相比，用于CD 2D凝胶检测的总灵敏度增加> 30倍）;（3）开发和测试在切割后留下小的同位素标签的可切割的巯基反应性Z染料，这将允许对每种感兴趣的蛋白质的相对量进行定量质谱分析，即使当两种或更多种蛋白质存在于靶向的凝胶斑点中时，（在细胞培养物中用氨基酸进行稳定同位素标记）来精确定位蛋白质以进行质谱分析-从而实现更有效的FET-SILAC实验;（4）开发和测试用于扩展的多通道复用的几种新的Zdyes颜色，以使得能够进行伴随例如多点剂量-响应曲线、多点动力学曲线的全局变化的单凝胶研究，并且同时，多个平行样本的全局比较;和（5）开发和测试用于Zdye和Z2 Dye设计的新的染料发色团框架，其将显著更容易和更便宜地生产，向最终用户提供更可负担得起的产品，可用于1D凝胶应用，并且将体现在第一代或第二代Zdyes中证明可行的相同设计概念。该项目具有很强的健康相关性，因为大部分蛋白质和蛋白质后修饰的异构体不能用当前的蛋白质组学技术可靠地检测。蛋白质翻译后修饰是大多数生物学机制的核心，因为翻译后修饰控制蛋白质活性、亚细胞定位和蛋白质-蛋白质相互作用。 公共卫生相关性：控制健康和疾病的细胞机制主要依赖于蛋白质和蛋白质的各种修饰来调节细胞活动，但目前的蛋白质组学技术仍然不足以可视化存在的所有蛋白质形式，并确定在生物反应期间发生变化的所有形式。该第二阶段STTR提案建立在第一阶段的成功基础上，以合成和展示一个新的不同颜色的高水溶性荧光染料（Zdyes）家族，该家族大大扩展了多重2D凝胶的独特能力，以揭示蛋白质相对量和蛋白质修饰的全球变化。Zdye技术提供的极大提高的灵敏度和大量样品条件的同时比较，将揭示蛋白质种类模式的变化，这些变化低至每个细胞单个蛋白质拷贝的极限，以更清楚地揭示生物机制的内部运作。

DESCRIPTION (provided by applicant): The broad long-term goals of this project are to develop more powerful proteomics detection technology with greatly improved sensitivity, that will reveal the global patterns of biologically stimulated changes (differential detection) in protein levels, posttranslational modifications and enzyme activities simultaneously (using 6-12 channel multicolor fluorescent dye (Zdye) detection), develop ways to use the patterns of protein isoform levels to recognize changing physiological states, and to more efficiently target and obtain the variable proteins for detailed mass spectral analysis. The specific aims and research design of the present proposal are to: (1) scale up the synthesis of amino-reactive Zdye and Z2dyes that were developed and shown feasible during the current Phase I proposal; (2) prepare and test highly water soluble Zdye saturation labels that react with protein thiols to avoid precipitation of labeled proteins (which is a problem with available thiol saturation labels for 2D gel detection), to facilitate spot picking or targeted electroelution of the proteins of each type in the saturation-labeled spots of interest, and to provide additional differential detection sensitivity, compared to Zdye amino labels (>30x total sensitivity increase compared to currently available amino labels for multicolor 2D gel detection); (3) develop and test cleavable thiol-reactive Zdyes that leave small isotopic tags behind after cleavage, which will allow quantitative mass spectral analysis of the relative amounts of each protein of interest, even when two or more proteins are present in the gel spots targeted, and demonstrate fluorescence enhanced targeted-SILAC (stable isotope labeling with amino acids in cell culture) to pin-point proteins for mass spectral analysis-- resulting in more efficient FET-SILAC experiments; (4) develop and test several new Zdyes colors for expanded multi-channel multiplexing, to enable single gel studies of global changes accompanying, for example, multi-point dose-response curves, multipoint kinetic curves, and simultaneous, global comparisons of several parallel samples; and (5) develop and test new dye chromophore frameworks for the Zdye and the Z2Dye designs that will be substantially easier and cheaper to produce, provide more affordable products to the end-user, may be feasible to use in 1D gel applications, and will embody the same design concepts proved feasible in the first or second generation Zdyes. This project has strong health-relatedness, since a large fraction of proteins and protein posttranslationally modified isoforms are not reliably detectable with current proteomics technology. Protein posttranslational modifications are at the heart of most biological mechanisms, since PTMs control protein activity, subcellular localization and protein-protein interactions. PUBLIC HEALTH RELEVANCE: Cellular mechanisms controlling health and disease depend primarily on proteins and a wide variety of modifications of proteins to regulate cellular activities, but current proteomics technology remains inadequate to visualize all the protein forms present and to determine all the forms that change during biological responses. This Phase II STTR proposal builds on the successful foundation of Phase I to synthesize and demonstrate a new family of different-colored, highly water soluble fluorescent dyes (Zdyes) that take greatly expanded advantage of the unique ability of multiplexed 2D gels to reveal global changes in the relative amounts of proteins and modifications of proteins. The greatly improved sensitivity and simultaneous comparison of a larger number of sample conditions, provided by the Zdye technology, will reveal the changes in the patterns of protein species that occur down to the limit of single protein copies per cell, to more clearly expose the inner workings of biological mechanisms.