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Author ORCID Identifier

N/A

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2016

Month Degree Awarded

February

First Advisor

Richard Vachet

Subject Categories

Analytical Chemistry | Biochemistry | Structural Biology

Abstract

Thorough insight into a protein’s structure is necessary to understand how it functions and what goes wrong when it malfunctions. The structure of proteins, however, is not easily analyzed. The analysis must take place under a narrow range of conditions or risk perturbing the very structure being probed. Furthermore, the wide diversity in size and chemistry possible in proteins significantly complicates this analysis. Despite this numerous methods have been developed in order to analyze protein structure. In this work, we demonstrate that mass spectrometry (MS)-based techniques are capable of characterizing the structure of particularly challenging proteins. This is done through the study of two model systems: (1) the amyloid forming protein β2-microglobulin and (2) the protein therapeutics human growth hormone and immunoglobulin G1. β-2-microglobulin (β2m) is an amyloidogenic protein and is the major constituent of fibrils in the disease dialysis related amyloidosis (DRA). Stoichiometric concentrations of Cu(II) have been used in vitro to induce the amyloid formation of β2m, but the structural changes caused by Cu(II) have not been fully elucidated. Other transition metals, such as Zn(II) and Ni(II), do not cause β2m amyloid formation, yet a comparison of the structural changes caused by these metals and those caused by Cu(II) could reveal essential structural changes necessary for amyloid formation. To probe these different structural changes, we have used a combination of hydrogen-deuterium exchange (HDX) and covalent labeling together with MS. Results from these measurements reveal that Cu(II) alone is capable of inducing the cis-trans isomerization of the X-Pro bond of Pro32 and the other necessary conformational changes that allow β2m to form an amyloid competent state, even though Ni(II) binds the protein at the same site. We also find that Zn(II) binding leads to increased dynamics, indicating increase structural instability, which is consistent with the amorphous aggregation observed in the presence of this metal. The second part of this dissertation investigates the use of diethylpyrocarbonate (DEPC)–based covalent labeling to detect three-dimensional structural changes in immunoglobulin G1 and human growth hormone after they have been exposed to degrading conditions. We demonstrate that DEPC labeling can identify both specific protein regions that mediate aggregation and those regions that undergo more subtle structural changes upon mishandling of these proteins. Importantly, DEPC labeling is able to provide information for up to 30% of the surface residues in a given protein, thereby providing excellent structural resolution. Given the simplicity of the DEPC labeling chemistry and the relatively straightforward mass spectral analysis of DEPC-labeled proteins, we expect this method should be amenable to a wide range of protein therapeutics and their different formulations. In the final section of this dissertation, we demonstrate that, in certain instances, scrambling of the DEPC label from one residue to another can occur during collision-induced dissociation (CID) of labeled peptide ions, resulting in ambiguity in label site identity. From a preliminary study of over 30 labeled peptides, we find that scrambling occurs in about 25% of the peptides and most commonly occurs when histidine residues are labeled. Moreover, this scrambling appears to occur more readily under non-mobile proton conditions, meaning that low-charge state peptide ions are more prone to this reaction. For all peptides, we find that scrambling does not occur during electron transfer dissociation, which suggests that this dissociation technique is a safe alternative to CID for correct label site identification.

DOI

https://doi.org/10.7275/7949898.0

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