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

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.