Development of Absolute Quantitative Protein Footprinting Mass Spectrometry (aqPFMS) for Probing Protein 3D Structures

开发用于探测蛋白质 3D 结构的绝对定量蛋白质足迹质谱 (aqPFMS)

• 批准号: 2203284
• 负责人: Hao Chen
• 金额: $ 39万
• 依托单位: New Jersey Institute of Technology
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2203284&HistoricalAwards=false
• 关键词: Development; Absolute; Quantitative; Protein; Footprinting

With support from Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry, Professor Hao Chen at the New Jersey Institute of Technology and collaborator Xiaolin Cheng from Ohio State University are working to develop and refine a novel approach called absolute quantitative protein footprinting mass spectrometry (aqPFMS) to probe protein 3D structures. Proteins play key roles as selective catalysts, receptors, antibodies and transporters in living systems, among other roles. Elucidation of dynamic protein three-dimensional (3D) structure in solution is highly challenging but critical for characterizing protein structure-function relationships at the molecular level. If successful, the quantitative protein footprinting MS tool under development here, in combination with protein modeling and simulation, can have high impact on structural biology and drug discovery. The team is engaging undergraduates in the research and reaching out to even younger students to acquaint them with this important interdisciplinary STEM (science, technology, engineering and mathematics) area.The aqPFMS technique is based on coupling a protein footprinting method with absolute peptide quantitation using coulometric mass spectrometry (CMS). Specifically, a footprinter reagent carrying an electrochemical tag reacts with proteins or protein complexes so that electrochemical oxidation can provide absolute quantitation of footprinted peptides resulting from enzymatic digestion of footprinted proteins without using normal standards. Combined with MS, the approach can provide quantitative analysis of solvent-accessible surface areas (SASAs) of different protein residues in any given conformation, thus providing greatly increased information for protein 3D structures. Structural modeling via all-atom molecular dynamics (MD) simulation can then be used to determine protein 3D conformations and conformational changes in solution with high confidence. If successful, this work has potential for broad scientific impact given the importance of protein dynamics and the challenge of experimentally capturing the three dimensional structures of multiple conformations of a single protein experimentally.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在化学系化学测量和成像（CMI）项目的支持下，新泽西理工学院的Hao Chen教授和来自俄亥俄州州立大学的合作者Xiaolin Cheng正在开发和改进一种称为绝对定量蛋白质足迹质谱（aqPFMS）的新方法来探测蛋白质3D结构。蛋白质在生命系统中起着选择性催化剂、受体、抗体和转运蛋白等关键作用。 阐明溶液中蛋白质的动态三维（3D）结构具有高度挑战性，但对于在分子水平上表征蛋白质结构与功能关系至关重要。 如果成功的话，这里正在开发的定量蛋白质足迹MS工具，结合蛋白质建模和模拟，可以对结构生物学和药物发现产生很大的影响。 该团队正在吸引本科生参与研究，并向更年轻的学生伸出援手，让他们熟悉这一重要的跨学科STEM（科学，技术，工程和数学）领域。aqPFMS技术基于将蛋白质足迹法与使用库仑质谱法（CMS）的绝对肽定量相结合。具体地，携带电化学标签的足迹试剂与蛋白质或蛋白质复合物反应，使得电化学氧化可以提供由足迹蛋白质的酶消化产生的足迹肽的绝对定量，而不使用正常标准。 结合MS，该方法可以提供定量分析的溶剂可及表面积（SASA）的不同蛋白质残基在任何给定的构象，从而大大增加了蛋白质的三维结构的信息。然后，通过全原子分子动力学（MD）模拟的结构建模可以用于以高置信度确定蛋白质3D构象和溶液中的构象变化。如果成功，这项工作有可能产生广泛的科学影响，因为蛋白质动力学的重要性和通过实验捕获单一蛋白质多种构象的三维结构的挑战。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。