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

项目总结 R&D项目2.分析阶段I：用于分析化学成分和化学计量的工具 大分子组装体 组装体内蛋白质的测定和定量仍然是一个巨大的挑战，因为蛋白质携带 细胞的快速动态反应的大部分负担，它们的丰富性和多样性令人难以置信 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。这项研究和发展将使研究人员能够定量地表征 隔离的程序集，并且只需通常所需的时间、精力和资源的一小部分即可实现这一目标。 因此，我们将开发方法，以忠实和充分地识别任何目标的骨骼成分 细胞中的大分子复合体，以及邻近缔合体的复合体，具有高准确度和高吞吐量。 我们将推进目前确定复杂组成的定量方法。我们还将提供 社区提供了一套区分特定交互作用者和污染物的选项。我们还将 开发方法，以高精度、准确地确定任何目标大分子的化学计量比 细胞内的复杂性。我们将以“化学计量学套餐”的形式向社会提供这项技术。 该包将包含一套并行的技术，以提供补充和交叉验证 信息，共同提供准确、全面的化学计量数据集(和动态变化) 用于独立的程序集。用户可以根据需要选择任何一种方法或方法的组合。我们会 还可以通过分离定义的络合物来收集有关高分子络合物动力学的数据 空间和时间序列；复合体如何随时间或不同组装状态变化的比较 在成分和化学计量方面，将允许重建这些动态过程。少校 我们在这里解决的挑战是以一种容易实现的模式以高保真的方式实现这些目标 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.