Author ORCID Identifier

https://orcid.org/0009-0005-2752-2591

Semester

Summer

Date of Graduation

2025

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Stephen Valentine

Committee Co-Chair

Peng Li

Committee Member

Hacer Karatas Bristow

Committee Member

Fabien Goulay

Committee Member

Liliya Yatsunyk

Abstract

A gentle or soft Ionization process is a critical component in native mass spectrometry (MS) to lift the biomolecules from the bulk solution into the gas phase environment of the mass spectrometer preserving their shape, topology and non-covalent interactions. Electrospray ionization (ESI) facilitates structural preservation of proteins, nucleic acids and their complexes and as a result it has been extensively used in native MS. However, in negative ion mode, ESI is somewhat limited in sensitivity due to corona discharge effect resulting from the requirement of a high amount of voltage. This often disrupts the ESI process and may not preserve the native conformations. To overcome such limitations, a novel spray-based, soft ionization called capillary vibrating spray ionization has been introduced which is simple to operate, doesn’t require nebulization gas and offers superior sensitivity for oligonucleotide analysis in negative ion mode. cVSSI features a wider operational range of applied voltage to the sample solution which allows the observation of folding/unfolding/refolding transitions of proteins and DNA. Another groundbreaking feature of cVSSI is that it can be easily coupled with ultrafast, in droplet hydrogen-deuterium exchange mass spectrometry (HDX-MS) in a dual emitter tip fashion. This unique HDX-MS method can differentiate co-existing conformational states of different types of DNA and proteins in a robust fashion. Another key feature of in-droplet HDX is that it reveals structural flexibility. Additionally, when combined with techniques such as molecular dynamics (MD) simulations it can suggest individual exchange sites of the biopolymers. A remarkable finding of this study is the distinguishing of linear vs globular forms of folded DNA species. In general, most globular G-quadruplex DNA structures protect exchange sites to a greater degree than the linear duplex DNA. Comparatively, another complex form of linear structure such as triplex DNA exhibits greater protection than duplex DNA even rivaling that of the most stable G-quadruplex structures. More interestingly, this in-droplet cVSSI-HDX-MS technique can capture subtle structural differences between different topologies and sub-topologies of globular G-quadruplex DNA. Currently, the differentiation has been ascribed to differences in conformer flexibility. In separate studies, the structural compactness of these therapeutic target G-quadruplexes is also susceptible to droplet charge tuning in the VSSI system in a conformer stability manner. Overall, structures remain in more unfolded or partially folded states in the low to mid voltage regions while more folded states predominate under high voltage settings. Observation of this folding/unfolding transition led to the development of a new method to study the stability of G-quadruplex DNA structure by changing (tuning) droplet charge using cVSSI. The elucidated in-droplet structural stability displays the following order: 23TAG

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