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

在化学系化学测量与成像（CMI）项目的支持下，新泽西理工学院的陈浩教授和俄亥俄州立大学的程晓林教授正在努力开发和完善一种称为绝对定量蛋白质足迹质谱（aqPFMS）的新方法，以探测蛋白质的3D结构。蛋白质在生命系统中扮演着选择性催化剂、受体、抗体和转运体等重要角色。阐明蛋白质在溶液中的动态三维结构是非常具有挑战性的，但对于在分子水平上表征蛋白质的结构-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质谱工具，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重大影响。该团队正在让本科生参与研究，并向更年轻的学生伸出援手，让他们熟悉这个重要的跨学科STEM（科学、技术、工程和数学）领域。aqPFMS技术是基于蛋白质足迹法与绝对肽定量的库仑质谱（CMS）耦合。具体来说，携带电化学标签的足迹试剂与蛋白质或蛋白质复合物发生反应，因此电化学氧化可以在不使用正常标准的情况下提供酶切足迹蛋白产生的足迹肽的绝对定量。结合质谱，该方法可以对任意给定构象的不同蛋白质残基的溶剂可及表面积（SASAs）进行定量分析，从而大大增加了蛋白质三维结构的信息。通过全原子分子动力学（MD）模拟的结构建模可以高可信度地用于确定蛋白质的三维构象和溶液中的构象变化。如果成功，考虑到蛋白质动力学的重要性和实验捕捉单个蛋白质多种构象的三维结构的挑战，这项工作具有广泛的科学影响的潜力。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.