Bush, Matthew F.Fawcett, Elizabeth Anne2025-10-022025-10-022025-10-022025Fawcett_washington_0250O_28469.pdfhttps://hdl.handle.net/1773/53939Thesis (Master's)--University of Washington, 2025Temperature-controlled electrospray ionization (tcESI) coupled with mass spectrometry (MS), which allows for the measurement of mass-to-charge (m/z) ratios, enables the analysis of both structural information and stability in proteins, specifically by evaluating their thermal denaturation, a measure of protein unfolding. In this work, programmed-temperature electrospray ionization (ptESI) source, is used to continuously and rapidly heat and cool a nanoESI capillary containing proteins in solution. Combining MS and ptESI, enables the tracking of structural changes in unfolding and refolding events from solution-phase chemistry. In ion mobility (IM) measurements, charged ions under the influence of an electric field experience collisions that allow for separation based on size, shape, and charge, which yields additional insights into protein structure. During collision-induced unfolding (CIU), collisional activation allows IM-MS to probe gas-phase ion structures regarding their stability. Shifts in stability, when assessing collisional activation, may be reflective of structural changes. In chapter 1 of this work, the tools traditionally used for assessing protein structure and stability are described. In chapter 2, the acquisition software updates, supporting usability and maintenance, for the ptESI source are characterized. Next, in chapter 3, the programmatic analysis of the structural and stability information generated using the acquisition software is described. Then, in chapter 4, experiments utilizing ptESI, CIU, and ptESI coupled with CIU are characterized. Finally, in chapter 5, an experiment utilizing ptESI coupled with collision-induced unfolding is introduced, describing the application of the acquisition software and the subsequent programmatic analysis. Overall, this work marks a significant programmatic milestone towards automating the collection of both MS and IM-MS data utilizing the ptESI source. This work represents significant milestones towards being able to screen protein libraries or protein-ligand libraries in drug discovery and development. Appendix A supplements Chapter 2 and contains a render of the physical ptESI source. Appendix B supplements Chapter 3 and contains examples of input and output data and visualizations related to the analytical pipeline. Appendix C supplements Chapter 4 and contains additional visualizations to assist with the characterization of ptESI, CIU, and ptESI coupled with CIU work.application/pdfen-USCC BY-SAacquisition softwareelectrospray ionizationion mobilitymass spectrometryAnalytical chemistryChemistryThesis