Molecular Structure Determination by Mass Spectrometry and Computational Modeling

项目摘要/摘要 结构生物学在现代分子生物科学中发挥着核心作用，使更大的 理解和操纵生物分子作用的新机制。然而，尽管有巨大的 用于生成高分辨率分子模型、生物分子大家族和 生物分子复合体在蛋白质结构数据库中的表现仍然很差，这是由于 目前用于探测哺乳动物组织内蛋白质结构的技术和方法很少。一种方法 它已被成功地用于定性研究其中几个家族的结构是羟基 自由基蛋白足迹(HRPF)，一种用于研究蛋白质变化的新兴技术 通过测量原位生成的羟基自由基间的表观反应速率的变化来测量地形 和蛋白质表面的氨基酸侧链。我们的初步工作已经将HRPF发展成一个量化的 在单个氨基酸水平上测量蛋白质的地形，精确地测量平均值 单一实验中许多氨基酸的溶剂可及表面积(&lt；sasa&gt；)。在这 更新，我们将把我们的技术扩展到随时间动态变化的结构系统，包括 蛋白质翻译后修饰系统、大的异构体蛋白质复合体和蛋白质：碳水化合物 复合体。我们将开发的实现这些研究的核心技术是高性能液体 联用氨基酸拆分HRPF(LC-HR-HRPF)。联用液相色谱 允许分离蛋白质构象并立即进行纯化的定量测量 在动力系统有机会重新平衡之前，通过HR-HRPF测量构象，冻结 稳定的化学足迹中的结构信息。我们还将开发蛋白质分析技术 哺乳动物全血中的结构，使研究蛋白质结构和相互作用成为可能 复杂的本机系统。我们将开发流动系统，以准确和仔细地将过氧化氢输送到 血液对蛋白质的标记不会破坏细胞，并将展示这项技术的结构 对注入小鼠模型的单抗进行分析。最后，我们将开发技术来探测 复杂碳水化合物的地形，使我们能够测量碳水化合物的哪些部分参与 与蛋白质的相互作用，即使在复杂的多糖混合物中也是如此。我们将开发特定于减速端的和 探测碳水化合物拓扑学的非特异性标记策略。总而言之，这些进步代表着 用于生物医学重要和极具挑战性的结构分析的潜在转换技术 系统。

PROJECT SUMMARY/ABSTRACT Structural biology plays a central role in modern molecular bioscience, enabling both a greater understanding and new mechanisms of manipulation of biomolecular action. However, despite tremendous development in tools for the generation of high resolution molecular models, large families of biomolecules and biomolecular complexes are still poorly represented in databases of protein structure due to limitations of current technology, and methods for probing protein structure within mammalian tissue are few. One method that has been used successfully to qualitatively study the structure of several of these families is hydroxyl radical protein footprinting (HRPF), an emerging technology that has been used to study changes in protein topography by measuring changes in the apparent rate of reaction between hydroxyl radicals generated in situ and amino acid side chains on the protein surface. Our initial work has developed HRPF into a quantitative measurement of protein topography at the individual amino acid level, accurately measuring the average solvent accessible surface areas (<SASA>) of many individual amino acids in a single experiment. In this renewal, we will expand our technology into structural systems that change dynamically with time, including protein posttranslational modification systems, large heteromeric protein complexes, and protein:carbohydrate complexes. The core technology we will develop to enable these studies is high performance liquid chromatography coupled inline with amino acid resolution HRPF (LC-HR-HRPF). Inline liquid chromatography allows the separation of protein conformers and immediate quantitative measurement of the purified conformers’ topographies by HR-HRPF before the dynamic system has a chance to re-equilibrate, freezing the structural information in the stable chemical footprint. We will also develop technology for analysis of protein structure within mammalian whole blood, enabling the study of protein structure and interactions within highly complex native systems. We will develop flow systems to precisely and carefully deliver hydrogen peroxide to blood for protein labeling without damaging cells, and will demonstrate the technology with the structural analysis of monoclonal antibodies dosed into a mouse model. Finally, we will develop technologies to probe the topography of complex carbohydrates, enabling us to measure which parts of carbohydrates mediate interactions with proteins, even in complex mixtures of glycans. We will develop both reducing-end specific and non-specific labeling strategies for probing carbohydrate topography. Together, these advances represent potential transforming technologies for the structural analysis of biomedically important and highly challenging systems.