Rapid Assessment of Proteoform-Resolved Higher-Order Structures

快速评估蛋白质型解析的高阶结构

• 批准号: 10714301
• 负责人: Nicholas Brent Borotto
• 金额: $ 35.92万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-20 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714301
• 关键词: Acceleration; Adoption; Automobile Driving; Biological; Cardiovascular Diseases; Cell physiology; Chemicals; Derivation procedure; Digestion; Disease; Distal; Ensure; Goals; Health; Higher Order Chromatin Structure; Human; Knowledge; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Methods; Modeling; Molecular Conformation; Nature; Neurodegenerative Disorders; Peptides; Post-Translational Protein Processing; Process; Proteins; Proteomics; Reagent; Regulation; Reporting; Reproducibility; Research Proposals; Sampling; Series; Spectrometry; System; Techniques; Therapeutic Intervention; complex biological systems; design; diagnostic value; drug development; drug discovery; improved; insight; ion mobility; new technology; new therapeutic target; protein function; tool

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.

项目描述 为了执行其多种功能，蛋白质通常会依次（顺序）将其置于和折叠 进入可再现的高阶结构（HOS）。确保这种方向或故意的完整性 通过翻译后修饰（PTM）调节它是已知被扰动的重要过程 在癌症，神经退行性和心血管疾病中。表征PTM如何改变蛋白质HOS可以 被用来识别疾病状态并为新目标治疗的设计提供信息和加速 干预措施。这是为了提高诊断能力，药物开发并提供有关 驱动细胞功能和疾病的基本机制，开发了能够 表征每种蛋白质存在的所有各种PTM调节的HOS都至关重要。这 灵敏度，高通量性质和质谱质量光谱法（MS）的样本要求较低 导致其采用作为测量蛋白质HOS和PTM的工具。不幸的是，员工工作流程最多， 自下而上的蛋白质组学要求分析物的蛋白水解消化，因此无法区分 从中得出任何单个肽的蛋白质类型。因此，自下而上的工作流报告平均 所有现有蛋白质成型的构象和所有PTM的平均占用率。这是为了精确评估 基于PTM的蛋白质功能调节，在接下来的五年中，我们建议开发新技术 那对夫妇自上而下 展开。这些新方法将使用良好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过程。除了这些生物学见解外，我们预计这是 研究建议将生成一系列基于MS的分析技术和各种化学技术 将适用于多种蛋白质系统的试剂。