Investigating structural and stability characteristics of native-like proteins using ion mobility-mass spectrometry (IM-MS) in positive- and negative-ion mode.

Native ion mobility-mass spectrometry (IMS-MS) is widely used to investigate the structure and stability of biologically relevant proteins and protein complexes. The key goal, and arguably biggest attraction, of native IMS-MS is maintaining the noncovalent interactions responsible for stabilizing the three-dimensional structures of a protein when it is introduced to the gas-phase. By maintaining and investigating a folded protein's gas-phase structure, one can learn the stoichiometry, subunit composition, and binding constants. The focus of this dissertation is investigating several fundamental aspects of native IMS-MS for analysis of protein and protein complexes. In Chapter One, the importance of studying protein structure and how native IMSMS is a valuable tool to do so is discussed in detail. In Chapter Two, we investigate the sources of uncertainty present in native IMS-MS analysis. There are several sources of uncertainty in IMS analysis that are not routinely monitored or regularly reported, particularly for Traveling-Wave Ion Mobility Spectrometry (TWIMS) calibration, so we developed an error propagation method that quantifies some of this uncertainty. Quantifying this uncertainty allows for a more robust comparison of protein gas-phase structures. Chapter Three focuses on how a protein's net-charge in solution affects its overall structure and stability in the gas-phase. We were able to demonstrate that some proteins are more stable in the gas-phase when analyzed in the ion mode matching their solution-phase charge, thus indicating the ionization process can cause changes to a protein's stability even when it does not change the overall three-dimensional structure. Lastly, in Chapter Four we use native IMS-MS to determine the structure and stability of a biologically relevant ternary enzymatic complex, highlighting the utility of native IMSMS for characterization of bound and unbound enzymatic samples. Together, this work broadens the native IMS-MS communities' understanding of several aspects of nativelike protein analysis via IMS-MS while also showcasing the utility of such techniques for the analysis of biologically relevant samples.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Doctoral

Grantor

Baylor University.

Year dc:date.issued

2023

Author dc:creator

• Edwards, Alexis N., 1997-

Advisor dc:contributor.advisor

• Gallagher, Elyssia S.

• Mass spectrometry.
• Native mass spectrometry.
• Collision induced unfolding.
• Ion mobility.

Statement dc:rights

• Baylor University works are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. Contact libraryquestions@baylor.edu for inquiries about permission.

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/2104/12859

OAI identifier oai:identifier

oai:baylor-ir.tdl.org:2104/12859