Advancing Mass Spectrometry Analyses of Proteins, Assemblies, and Proteoforms

项目摘要/摘要 深入了解细胞过程和途径需要的方法不仅是研究 各种蛋白质组合的组成和排列，但这也阐明了它们的动力学和 功能调控。传递主序列以外的结构信息的实验策略；即， 高阶结构和蛋白质修饰，将蛋白质组学与结构生物学联系起来 对复杂疾病机制的潜在见解。基于质谱学的新技术， 原生MS(即，测量保留配体的原生溶液环境中的生物分子--以及其他 分子相互作用)，以及自上而下的MS/蛋白质组学将被推进和应用于促进 蛋白质和蛋白质组件的表征。灵敏电离、溶液和气相的耦合 分离，新的基于电子的解离方法(例如，电子捕获解离，ECD；电子 电离解离(EID)和其他激活/解离技术来获得超高分辨率质量 光谱分析将为全序列和蛋白质形式覆盖提供一个实验平台。这些 将使用先进的工具来表征重要的生物络合物，其高分辨率 结构一直很难获得，包括G偶联蛋白受体(GPCRs)和其他膜 蛋白质。翻译后修饰(PTM)与蛋白质形式之间联系的探索 阿尔茨海默病(AD)等神经退行性疾病的聚集和毒性将通过 我们先进的质谱学方法。我们的实验策略应用广泛，并与众多 生物物理技术使大型和复杂分子机器的详细结构研究成为可能，并 提供对其动态的洞察。自然自上而下的多发性硬化症承诺推进结构生物学并加快 药物发现和开发。基于MS的技术的改进可以促进我们对 蛋白质和蛋白质机器如何推动生物学。

PROJECT SUMMARY/ABSTRACT Understanding cellular processes and pathways in depth requires methods that not only investigate the composition and arrangement of various protein assemblies, but that also elucidate their dynamics and functional regulation. Experimental strategies delivering structural information beyond primary sequence; i.e., higher-order structure and protein modifications, connect proteomics to structural biology in ways yielding potential insights into complex disease mechanisms. New techniques based on mass spectrometry (MS), native MS (i.e., measuring biomolecules in their native solution environment preserving ligand- and other molecular interactions), and top-down MS/proteomics will be advanced and applied to facilitate the characterization of proteins and protein assemblies. Coupling sensitive ionization, solution- and gas-phase separations, new electron-based dissociation methods (e.g., electron capture dissociation, ECD; electron ionization dissociation, EID) and other activation/dissociation techniques to ultra-high resolution mass spectrometry will provide an experimental platform for complete sequence and proteoform coverage. These advanced tools will be employed to characterize important biological complexes for which high-resolution structures have been difficult to obtain, including G-coupled protein receptors (GPCRs) and other membrane proteins. The exploration of links between post-translational modifications (PTMs) and proteoforms to the aggregation and toxicity of neurodegenerative diseases such as Alzheimer's disease (AD) will be furthered by our advanced MS methods. Our experimental strategies apply broadly and integrate with numerous biophysical techniques to enable the detailed structural study of large and complex molecular machines and to provide insights into their dynamics. Native top-down MS promises to advance structural biology and to hasten drug discovery and development. Improvements in MS-based technologies can advance our understanding of how proteins and protein machines drive biology.