Author ORCID Identifier

https://orcid.org/0000-0001-8436-8088

Date of Award

8-31-2026

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry/Biological Chemistry

First Advisor

Jason Evans

Second Advisor

Michael J. Eck

Abstract

Mass spectrometry has become an essential analytical platform for the characterization of complex biological systems, enabling detailed investigation of molecular structure, post-translational modifications, and protein expression. This dissertation applies modern mass spectrometry methodologies to three analytical challenges spanning lipidomics and proteomics.

The first study examined the influence of complexing agents on the sensitivity of collision-induced dissociation (CID) spectra to fatty acid position in synthetic XYZ-type triglycerides. Comparison of ammonium, lithium, and sodium adducts demonstrated that alkali metal adducts provide substantially improved positional sensitivity and greater consistency across triglyceride systems. These findings support the use of lithium and sodium adducts for structural characterization of triglycerides by tandem mass spectrometry.

The second study resulted in a quantitative phosphoproteomics workflow for determining phosphorylation site occupancies and activation stoichiometries within the EGFR/MAPK signaling pathway. By combining tandem mass tag (TMT) labeling, phosphopeptide enrichment, targeted SPS-MS3 acquisition, and discovery proteomics, time-resolved phosphorylation dynamics were quantified across multiple cellular models. This approach enabled simultaneous pathway-focused and proteome-wide assessment of phosphorylation regulation.

The final study evaluated the feasibility of absolute quantification of MAPK pathway proteins using stable isotope-labeled AQUA peptides and trapped ion mobility spectrometry coupled with parallel reaction monitoring (PRM-PASEF). Method development focused on optimization of peptide selection, sample preparation, chromatographic separation, and ion mobility acquisition parameters. Although the optimized workflow provided robust detection and quantification of isotopically labeled AQUA standards, endogenous target peptides remained below the limits of reliable quantification under the conditions tested, highlighting key analytical challenges and providing a framework for future assay development.

These studies highlight the versatility of mass spectrometry for structural lipidomics, quantitative phosphoproteomics, and targeted proteomics while providing methodological advances that improve the characterization of molecular composition, signaling regulation, and protein abundance in biological systems.

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