Development and Application of Mass Spectrometry-Based Protein Footprinting in Structural Proteomics
Date of Award
Doctor of Philosophy (PhD)
Integral mass spectrometry (MS) has emerged as an important tool for protein structural characterization. It readouts are a broad range of structural information, including stoichiometry, interactions, conformations and conformation change, and dynamics. Protein footprinting is a pivotal component in the intergral MS toolkit.My dissertation centers around the development and application of protein footprinting to characterize protein structure. It is divided into seven chapters.Chapter 1 serves as the introduction for integral mass spectrometry in structural proteomic.In Chapter 2, we extended the fast-photochemical oxidation of proteins (FPOP) platform by adding the trifluoromethyl radical (•CF3) as a new reagent. We discovered that •CF3 footprint proteins in a complementary way as hydroxide radicals. The •CF3 radical has exceptional reactivity, modifying 18 amino acids out of 20. Further studies demonstrate that it can report the conformational change between holo-myoglobin and apo-myoglobin and can define the topology of the VKOR membrane protein. This work bridges trifluoromethylation chemistry in materials and medicinal chemistry to that in structural biology.In Chapter 3, collaborated with Dr. Mark Chance’s laboratory in Case Western Reserve University (CWRU) to apply •CF3 chemistry on the synchrotron platform, which is the first platform that uses •OH for protein footprinting. Synchrotron radiolysis generates •CF3 in water media by ionizing water molecules to give •OH. The •CF3 shows complementary chemical reactivity with canonical •OH labeling yet results in higher reactivity coverage. The •CF3 reagent is the second footprinting reagent enabled by synchrotron since 1999. This work serves as a proof-of-concept to demonstrate that synchrotron platform is adaptable to other novel chemistries that can increase footprinting coverage. Further, taking advantage of X-ray irradiation, we achieved direct protein trifluoromethylation in the absence of metal catalysis or peroxide for the first time, with the synchrotron platform.In Chapter 4, we devloped a laser-mediated radical method for integral membrane protein (IMP) footprinting. Classical footprinting methods use hydrophilic reagents to label protein surfaces. IN so doing, we generate structural information by measuring the solvent accessibility of the backbone or side chains in aqueous media. Owing to the amphipathic nature of IMP, this new approach exploits the highly hydrophobic nature of perfluoroalkyl iodine together with tip sonication to ensure efficient labeling of a transmembrane domain (TM). The chemistry yields 100% reactivity coverage for tyrosine, and complete IMP labeling in a fast fashion. The resulting protein modification, which is resistant to hydrolysis, compatible with proteolysis, and amenable of tandem mass analysis, is appropriate for footprinting by bottom-up analysis. (Collaboration with Dr. Weikai Li from Wash U Medical School)In Chapter 5, we investigated an array of digestion conditions by using different combination of protease and additives to optimize the coverage of IMP digestion. IMPs are under-represented in conventional bottom-up proteomic analysis that generally favors soluble, abundant and easy-to-digest proteins. The new protrocol of IMP digestion significantly decreases our workload for sample preparation, allows us to avoid common contaminants that impair LC-MS, and generally yields >90% sequence coverage by generating peptides suitable for structural proteomic studies. Further, the deep analysis enable us to identify a “sweet spot” in the digestion protocol that may provide guidance to choose a suitable protease in structural proteomics in future.In Chapter 6, apart from methodology development, we used hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize the binding interface for Mxra8-immune complex and Mxra8-chikungunya virus protein complex. The cell adhesion molecule Mxra8 is identified as a receptor for multiple arthritogenic alphaviruses such as chikungunya virus. We identified putative binding sites for eight anti-Mxra8 monoclonal antibodies (mAbs). HDX-MS enables us to classify the novel mAbs, predict their competing binding interface with chikungunya virus, and provide a molecular level explanation for the observation that mAbs can block the Chikungunya virus infection. From the HDX kinetic curves, we also observe that the mAbs have higher affinity than do Chikungunya virus proteins when binding with Mxa8. Finally, the HDX data help to assign the orientation of Mxra8 on the Cryo-EM structure of Chikungunya virus complex (Collaboration with Dr. Daved H. Fremont and Dr. Michael s Diamond, Wash U School of Medicine).In Chapter 7, we provide a conclusion for my dissertation. We will discuss challenges and opportunities for protein footprinting, and its role in the expanding toolkit of structural proteomics.
Chair and Committee
Michael L. Gross
Daved H. Fremont, Meredith Jackrel, Weikai Li, John-Stephen Taylor,
Cheng, Ming, "Development and Application of Mass Spectrometry-Based Protein Footprinting in Structural Proteomics" (2019). Arts & Sciences Electronic Theses and Dissertations. 1995.
Permanent URL: https://doi.org/10.7936/aztc-zt80