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Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Richard W. Vachet
This dissertation focuses on the mass spectrometric based methods for studying protein structures in solution phase and in gas phase. Protein structure is important to study because it is related to many disease mechanism and related therapies. The relationship between protein structures in solution phase and in gas phase has been investigated. Especially, the electrostatic interaction is characterized by mass spectrometry (MS). A HDX method has been developed to study protein oligomer kinetics and applied to study β-2-microglobulin (β2m) oligomerization.
This dissertation firstly focuses on the relationship between protein gas phase structure and solution phase structure. We studied the structures of gas phase protein ions electrosprayed from native solution by combining Electron transfer dissociation (ETD) and collision induced dissociation (CID) in different orders. ETD together with ETD/CID was used to support the idea that electrostatic interactions that exist in solution can be maintained in gas phase protein ions. 4 of 5 studied dissociate in ETD and ETD/CID in a way that is consistent with the pattern of salt bridges known from solution structures. Beyond that, the stabilities of the electrostatic interactions was studied in 3 proteins. Collisional activation (CA) was performed before ETD to implement CaETD. We provide evidence that stabilities of salt bridges could differ from each other. The number of salt bridges that are broken, higher gas-phase basicities, and presence of nearby charged residues could be factors to influence the energy that is needed to disrupt the salt bridge.
The rest of this dissertation focuses on study protein structure and protein complex kinetics by hydrogen deuterium exchange (HDX). We began with developing an innovative HDX methodology to study both the structure and oligomer association/dissociation rate simultaneously. Beta-lactoglobulin (βLG) was used as a paradigm protein. Not only the βLG dimer structure was able to be characterized, the dimer’s dissociation rate can also be measured. The method has been validated by comparing data to the known protein structure and reported dissociation rates. This method was then applied to an amyloid formation protein, beta-2-microglobulin (β2m). The Cu(II)-induced dimer was firstly studied. Three strands were found to involve dimer interface. The dissociation rate was also measured and compared to the value under a lower ionic strength. The importance of the electrostatic interaction was therefore revealed. The Zn(II)-induced dimer was also studied. There was one strand and part of a loop found to involve dimer formation by HDX-MS. The difference in structures of two dimers shows a positive correlation between number of residues involving dimer interface and the likelihood of amyloid fibrils formation. In addition, the Zn(II)-induced dimer was found to dissociate much slower than Cu(II)-induced dimer. This provides an insight of the feature for proteins to form amyloids that the fast-exchanging kinetics between oligomer species could be the feature that allows oligomers to form amyloid fibrils.
Zhang, Zhe, "New Mass Spectrometric Methods for Studying Proteins in Solution and in the Gas Phase" (2017). Doctoral Dissertations. 907.