Author ORCID Identifier

https://orcid.org/0000-0001-8397-0536

Semester

Summer

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Stephen Valentine

Committee Member

Glen Jackson

Committee Member

Peng Li

Committee Member

Carsten Milsmann

Committee Member

Patrick Callery

Abstract

Biopolymers, such as proteins and nucleic acids, serve important functions dictated by their structural folding. Information excavated from the intricate structural arrangements of biopolymers have been proved to be vital in diagnosing diseases, unveiling disease mechanisms, and developing new drugs. A continuous effort has been put to improve the characterization techniques of these biomolecules so that more precision can be achieved in terms of detailing their structure-function relationship. Mass spectrometry (MS) is one such technique that has become very relevant for structural biology with the emergence of soft ionization techniques such as electrospray ionization (ESI). A prerequisite to the application of ESI-MS for structural investigation of biopolymers is that the tertiary or quaternary structural components need to be retained in the gas phase of MS. The subdivision of biological mass spectrometry that employs techniques and practices to preserve the native structures of biomolecules in the gas phase is now largely known as ‘native MS’. With the growing complexity of the targeted analytes, native MS promotes the inclusion of techniques that are more capable of deciphering the dynamic structural behavior of biopolymers. For example, proteins that are of intractable (very flexible) nature or that exist in multiple conformations in solution, require an MS-based approach beyond regular ESI to address these challenges. A new spray-based ionization technique namely capillary Vibrating Sharp-edge Spray Ionization (cVSSI) is demonstrated to provide improved field-free ionization capability to promote conformer preservation in the gas phase of MS, especially for the proteins possessing greater conformational flexibility in solution. Two such proteins, leptin and thioredoxin, exhibit bimodal charge state distributions (CSDs) upon the application of voltage; only a monomodal distribution is observed for field-free conditions of cVSSI. In addition, a multidevice cVSSI approach is demonstrated to achieve in-droplet hydrogen/deuterium exchange to directly probe the solution conformer heterogeneity of a well-studied protein, ubiquitin. The presence of co-existing protein solution structures under native and denaturing solution conditions have been distinguished by the isotopic distributions of deuterated ubiquitin ions. The interchange of the relative presence of the conformers within the ensembles corresponds accordingly with the degree of denaturants (methanol) added. Finally, cVSSI is demonstrated as an ionization platform to perform controlled unfolding/refolding of ubiquitin in the droplets by adding voltage sequentially. The mass spectral features are monitored carefully to detail the impacts of voltage related to structural transformations, adducts, and multimers of ubiquitin.

Embargo Reason

Publication Pending

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