Rapid Assessment of Proteoform-Resolved Higher-Order Structures

项目说明 为了执行它们的多种功能，蛋白质通常将序列末端的残基聚集在一起并折叠。 形成可复制的高阶结构(HOS)。确保此方向的完整性或故意 通过翻译后修饰(PTM)对其进行调节是已知受到干扰的重要过程 癌症、神经退行性疾病和心血管疾病。PTM如何改变蛋白质HOS的特征 用于识别疾病状态，通知并加速新的靶向治疗方案的设计 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 确定每种蛋白质都存在的各种不同的PTM调节的HOSS是至关重要的。这个 质谱学具有灵敏度高、高通量、样品要求低等特点。 导致它被用作测量蛋白质HOS和PTM的工具。不幸的是，使用最多的工作流程， 自下而上的蛋白质组学，需要对分析物进行蛋白水解性消化，因此不可能区分 一种蛋白质形式，任何一种多肽都是从它衍生而来的。因此，自下而上的工作流会报告平均值 所有现有蛋白质的构象和所有PTM的平均占有率。因此，要准确地评估 基于PTM的蛋白质功能调控，我们建议在未来五年内开发新技术 这结合了自上而下的蛋白质组学、离子迁移率光谱、化学衍生化和碰撞诱导 正在展开。这些新方法将在开发之前使用具有良好特性的模型蛋白质系统 应用于更复杂的生物系统。这些技术的结合将提供重要的 洞察这些关键的监管流程。除了这些生物学的洞察力，我们预计这一点 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 将适用于多种蛋白质体系的试剂。

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.