Rapid Assessment of Proteoform-Resolved Higher-Order Structures

项目描述 为了完成它们的多种功能，蛋白质经常将序列中的远端残基聚集在一起并折叠 可再现的高阶结构（HOS）。确保这一方向的完整性或故意 通过翻译后修饰（PTM）来调节它是已知会受到干扰的重要过程 癌症、神经退行性疾病和心血管疾病。表征PTM如何改变蛋白质HOS可以 用于识别疾病状态，并为新的靶向治疗提供信息和加速设计 干预措施。因此，为了提高诊断能力，药物开发，并提供对 驱动细胞功能和疾病的基本机制，开发能够 表征每种蛋白质存在的所有各种PTM调节的HOS是至关重要的。的 质谱（MS）的灵敏度、高通量性质和低样品要求已经越来越多地 导致其被采用作为测量蛋白质HOS和PTM的工具。不幸的是，最常用的工作流程， 自下而上的蛋白质组学，需要分析物的蛋白水解消化，因此，不可能区分 任何单一肽的蛋白形式。因此，自下而上的工作流报告平均值 所有现有蛋白质型的构象和所有PTM的平均占有率。因此，为了准确评估 基于PTM的蛋白质功能调控，在未来五年内，我们建议开发新技术 结合自上而下的蛋白质组学、离子迁移谱、化学衍生和碰撞诱导 展开这些新的方法将使用充分表征的模型蛋白质系统开发， 应用于更复杂的生物系统。这些技术的结合将提供重要的 深入了解这些关键的监管流程。除了这些生物学的见解，我们预计， 一项研究计划将产生一系列基于MS的分析技术和各种化学物质， 这些试剂将适用于广泛的蛋白质系统。

Project Description To perform their multitude of functions, proteins often bring—distal in sequence—residues together and fold into reproducible higher-order structures (HOS). Ensuring the integrity of this orientation or intentionally modulating it through post-translational modification (PTM) are vital processes that are known to be perturbed in cancers, neurodegenerative, and cardiovascular diseases. Characterizing how PTM alters protein HOS can be employed to identify disease state and inform and accelerate the design of new targeted therapeutic interventions. Thus, to improve diagnostic capabilities, drug development, and provide insight into the fundamental mechanisms driving cellular function and disease, developing techniques capable of characterizing all the various PTM-modulated HOSs that exist for each protein is vitally important. The sensitivity, high-throughput nature, and low sample requirements of mass spectrometry (MS) has increasingly led to its adoption as a tool to measure protein HOS and PTM. Unfortunately, the most employed workflow, bottom-up proteomics, demands proteolytic digestion of the analyte and thus, it is not possible to distinguish the proteoform from which any single peptide is derived. Therefore, bottom-up workflows report the average conformation of all existing proteoforms and the average occupancy of all PTMs. Thus, to precisely assess the PTM-based regulation of protein function, over the next five years, we propose to develop new technologies that couple top-down proteomics, ion mobility spectrometry, chemical derivatization, and collision-induced unfolding. These new methods will be developed using well characterized model protein systems prior to application to more complex biological systems. The combination of these techniques will offer significant insight into these critical regulatory processes. In addition to these biological insights, we anticipate that this research proposal will generate a series of MS-based analytical techniques and an assortment of chemical reagents that will be applicable to a wide range of protein systems.