TR&D Project 2. The Analysis Stage I: Tools for Analyzing the Composition and Stoichiometry of Macromolecular Assemblies

项目摘要 TR&D项目2.分析阶段I：用于分析有机化合物的组成和化学计量的工具 大分子组装体 组装体中蛋白质的测定和定量仍然是一个重大的挑战，因为蛋白质携带 大部分的负担的快速动态反应的细胞，是令人难以置信的多样性， 它们的物理化学性质，使它们对于手头的动态任务具有高度的通用性，但同时 时间很难分析。该TR&D将允许研究人员定量表征 独立的组件，并实现这一目标的一小部分的时间，精力和资源通常需要。 因此，我们将开发方法，以忠实地和充分地确定任何目标的真正成分 细胞内大分子复合物以及邻位缔合物，具有高精度和高通量。 我们将推进目前的定量方法来确定复杂的组成。我们亦会提供 社区与一套选择之间的具体相互作用和污染物的区别。我们还将 开发方法，以高精度忠实地和全面地鉴定任何目标大分子的化学计量 细胞中的复杂。我们会以“化学计量套件”的形式向社会提供这项技术。 该软件包将包含一套并行的技术，提供互补和交叉验证 信息，共同提供准确，全面的化学计量数据集（和动态变化） 一个孤立的集合。用户可以根据需要选择任何方法或方法组合。我们将 还收集数据，告知大分子复合物的动力学，通过分离复合物在定义的 空间和时间序列;比较复合物如何随时间或不同组装状态变化 将允许重建这些动态过程。主要 我们在这里所面临的挑战是以一种容易实现的模式高保真地实现这些目标。 任何研究人员都可以使用。

PROJECT SUMMARY TR&D Project 2. The Analysis Stage I: Tools for Analyzing the Composition and Stoichiometry of Macromolecular Assemblies The determination and quantitation of proteins within assemblies remain a significant challenge, as proteins carry much of the burden of the rapid dynamic responses of the cell and are incredibly diverse in their abundance and their physico-chemical properties, making them highly versatile for the dynamic tasks at hand but at the same time difficult to analyze. This TR&D will allow the researcher to quantitatively characterize the constitution of isolated assemblies, and achieve this objective with a fraction of the time, effort, and resources normally required. Thus, we will develop methods to faithfully and fully identify the bone fide components of any target macromolecular complex in the cell, as well as that of vicinal associators, with high accuracy and throughput. We will advance current quantitative approaches for determining complex composition. We will also provide the community with a suite of options for discriminating between specific interactors and contaminants.We will also develop methods to faithfully and fully identify with high accuracy the stoichiometry of any target macromolecular complex in the cell. We will provide this technology to the community in the form of a “Stoichiometry Package”. This package will incorporate a parallel suite of techniques that give complementary and cross-verifying information, collectively providing accurate, comprehensive sets of stoichiometry data (and dynamic changes) for an isolated assembly. Users can choose any or a combination of approaches, depending on need. We will also gather data that inform on the dynamics of macromolecular complexes, by isolating complexes in defined sequences in space and time; comparisons of how the complexes change with time or different assembly state in terms of composition and stoichiometry will allow reconstruction of these dynamic processes. The major challenge we are addressing here is to achieve these goals with high fidelity in a mode that is readily transportable to any researcher.