Molecular Structure Determination by Mass Spectrometry and Computational Modeling

项目总结/摘要 结构生物学在现代分子生物科学中起着核心作用， 理解和操纵生物分子作用的新机制。尽管巨大的 开发用于生成高分辨率分子模型、大家族生物分子和 生物分子复合物在蛋白质结构数据库中的表现仍然很差， 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 已经成功地用于定性研究这些家族中的几个家族的结构的是羟基 自由基蛋白质足迹（HRPF），一种新兴的技术，已被用于研究蛋白质的变化， 通过测量原位产生的羟基自由基之间的表观反应速率的变化来测量形貌 和蛋白质表面上的氨基酸侧链。我们的初步工作已经发展HRPF成为一个定量的 在单个氨基酸水平上测量蛋白质形貌，准确测量平均 溶剂可及表面积（<SASA>）的许多个别氨基酸在一个单一的实验。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、大异聚蛋白质复合物和蛋白质：碳水化合物 配合物我们将开发的核心技术，使这些研究是高性能液体 色谱法与氨基酸分辨率HRPF（LC-HR-HRPF）联用。在线液相色谱 允许蛋白质构象异构体的分离和纯化的构象异构体的立即定量测量。 在动态系统有机会重新平衡之前， 稳定化学足迹中的结构信息。我们还将开发蛋白质分析技术， 哺乳动物全血中的蛋白质结构，使研究蛋白质结构和相互作用的高度 复杂的原生系统我们将开发流动系统，以精确和小心地将过氧化氢输送到 血液的蛋白质标记，而不损害细胞，并将展示与结构的技术， 分析给药到小鼠模型中的单克隆抗体。最后，我们将开发技术， 复杂碳水化合物的拓扑结构，使我们能够测量碳水化合物的哪些部分介导 与蛋白质的相互作用，即使是在复杂的聚糖混合物中。我们将开发减少端特定和 用于探测碳水化合物形貌的非特异性标记策略。这些进步共同代表了 具有生物医学重要性和高度挑战性的结构分析的潜在转化技术 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.