Multiplex Fluorescent Dyes for Ultrasensitive Proteomics

描述（由申请人提供）：该项目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，揭示蛋白质水平、翻译后修饰和酶活性同时发生的生物刺激变化的全局模式（差异检测）（使用6-12通道多色荧光染料（Zdye）检测），开发利用蛋白质同工异构体水平模式识别生理状态变化的方法。更有效地定位和获取可变蛋白进行详细的质谱分析。本提案的具体目标和研究设计是：(1)扩大合成氨基反应性Zdye和z2染料的规模，这些染料在当前的第一阶段提案中已经开发并证明是可行的；(2)制备和测试与蛋白质硫醇反应的高水溶性Zdye饱和标签，以避免标记蛋白质的沉淀（这是二维凝胶检测中可用的硫醇饱和标签的问题），以便于在饱和标记的感兴趣的点上对每种类型的蛋白质进行点选取或靶向电洗脱，并提供额外的差分检测灵敏度。与Zdye氨基标签相比（与目前可用的多色二维凝胶检测氨基标签相比，总灵敏度提高了30倍）；(3)开发和测试可切割的硫醇反应性zdye，在切割后留下小的同位素标签，这将允许对每个感兴趣的蛋白质的相对数量进行定量质谱分析，即使在靶向凝胶点中存在两种或更多蛋白质，并展示荧光增强的靶向silac（细胞培养中氨基酸的稳定同位素标记）以精确定位蛋白质进行质谱分析-导致更有效的FET-SILAC实验；(4)开发和测试几种新的zdye颜色，用于扩展多通道复用，以实现伴随全局变化的单凝胶研究，例如多点剂量响应曲线，多点动力学曲线，以及同时，多个平行样品的全局比较；(5)为Zdye和Z2Dye设计开发和测试新的染料发色团框架，这些框架将更容易和更便宜地生产，为最终用户提供更实惠的产品，可能适用于1D凝胶应用，并将体现在第一代或第二代Zdye中被证明可行的相同设计概念。该项目具有很强的健康相关性，因为目前的蛋白质组学技术无法可靠地检测到大部分蛋白质和蛋白质翻译后修饰的同种异构体。蛋白质翻译后修饰是大多数生物学机制的核心，因为PTMs控制着蛋白质活性、亚细胞定位和蛋白质-蛋白质相互作用。

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.