Advancing Mass Spectrometry Analyses of Proteins, Assemblies, and Proteoforms

项目总结/摘要 深入了解细胞过程和途径需要的方法不仅要研究细胞的功能， 各种蛋白质组装的组成和排列，但也阐明了它们的动力学， 功能调节。实验策略传递结构信息超出一级序列;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。基于质谱分析（MS）的新技术， 原生MS（即，测量生物分子在其天然溶液环境中的保留配体-和其他 分子相互作用）和自上而下的MS/蛋白质组学将被推进和应用，以促进 蛋白质和蛋白质组装的表征。耦合灵敏电离，溶液和气相 分离，新的基于电子的解离方法（例如，电子捕获解离 电离解离，EID）和其它活化/解离技术来获得超高分辨率质量 光谱法将提供一个实验平台，完整的序列和proteoform覆盖。这些 将采用先进的工具来表征重要的生物复合体， 已经很难获得包括G偶联蛋白受体（GPCR）和其它膜受体的结构。 proteins.探索翻译后修饰（PTM）和蛋白质组之间的联系， 神经退行性疾病如阿尔茨海默病（AD）的聚集和毒性将进一步通过 我们先进的MS方法我们的实验策略应用广泛，并与许多 生物物理技术，使大型和复杂的分子机器的详细结构研究， 深入了解他们的动态。原生自上而下的MS有望推进结构生物学， 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是如何驱动生物的

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.