Semester

Spring

Date of Graduation

2026

Document Type

Dissertation (Campus Access)

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Fabien Goulay

Committee Co-Chair

Peng Li

Committee Member

Stephen Valentine

Committee Member

Glen Jackson

Committee Member

Tatiana Trejos

Abstract

Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI) is an ambient ionization technique that produces a plume of droplets through the vibration of a sharp-edge emitter via an amplified signal produced by a waveform generator and amplifier. The sample biomolecule within the plume can be analyzed by a mass spectrometer in both voltage-free and high voltage conditions. Already, cVSSI has shown that it has higher detection limits of biomolecules over that of electrospray ionization (ESI) in both positive and negative ion modes. However, cVSSI has the potential to be broadened for use in other applications besides the direct injection of a sample containing one biomolecule. In these studies, we aimed to utilize both instrument-based and solution-based methods to use in conjunction with or further enhance the cVSSI source itself such as adding supercharging reagents to enhance signal under negative ion mode, using applied amplitude voltage to tune droplets to smaller diameters, and combining our source with a microfluidic herringbone to study hydrogen-deuterium exchange reactions. Chapter 2 describes the use of a microfluidic herringbone 3D-printed device with cVSSI to study hydrogen-deuterium exchange (HDX) reaction kinetics. This device included inlets for a protein sample, deuterium, and quench. The results of this study showed the progression of exchange from 7 s to 70 s for a sample of 5 μM Myoglobin in 10 mM NH4OAc. Fast reaction time coincides with a lower amount of exchange occurring, while slow reaction time coincides with a higher amount of exchange occurring. Chapter 3 depicts the development of a technique to decrease the droplet size produced by a cVSSI source. Having the capability to fine-tune our ionization source to produce smaller droplet diameters is key to the preservation of the native conformation of a biomolecule. In this study, we showed that droplet diameters produced can be decreased by increasing the onset voltage, or amplitude, applied by our waveform generator. Droplets produced at different onset voltages were captured via brightfield microscopy to measure the diameters. A variety of biomolecules were tested as mass spectrometric models to depict the change in charge state distributions when decreasing the droplet size. Chapter 4 shows the potential to further enhance the signal under the negative ion mode. While cVSSI already shows improved signal over ESI, there are still biomolecules that struggle to ionize under negative ion mode, even with cVSSI. For this study, we investigated the effect supercharging reagents have on proteins and peptides when coupled with cVSSI ionization under positive and negative ion modes. Many acidic peptides show signal enhancement when 1% meta-nitrobenzyl alcohol (m-NBA) is added to the sample. Chapter 5 discusses the future directions of these projects and one potential study that was stumbled upon. A mass spectrometric model is still needed for the droplet generation project, and more investigation is needed to better understand what causes the droplet diameter decrease, as not every cVSSI device behaves the same. While many acidic peptides showed that adding 1% m-NBA to the solution enhanced signal intensity, some acidic peptides did not, and it is important to determine the mechanism behind this phenomenon. Finally, during the herringbone study, the Glucagon-Like Peptide-2 (GLP-2) was initially investigated and determined to have a dimer formation at low concentrations and was reduced at higher concentrations. It would be valuable to study this peptide further to understand its functionality as it is used for the treatment of various intestinal diseases.

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Available for download on Tuesday, April 27, 2027

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