Bush, Matthew FMartynova, Alice2026-04-202026-04-202026Martynova_washington_0250E_29309.pdfhttps://hdl.handle.net/1773/55460Thesis (Ph.D.)--University of Washington, 2026This dissertation describes new mass spectrometry (MS) and ion mobility-mass spectrometry (IM-MS) methodologies and fundamental investigations aimed at enhancing molecular characterization across diverse chemical systems. Mass spectrometry is a powerful analytical technique that measures ions' mass-to-charge (m/z) ratios, enabling determination of molecular mass and elemental composition. The structural information obtainable from MS can be further enhanced by coupling with ion mobility (IM), a gas-phase separation technique in which charged ions are separated based on their size, shape, and charge. From IM measurements, ions' collision cross-sections (CCS or Ω) can be determined, providing an additional dimension of structural information beyond mass alone. The work presented herein advances the quantitative assessment of IM data, improves calibration strategies for Traveling Wave Ion Mobility (TWIM) instruments, investigates fundamental electrospray ionization processes, demonstrates the utility of native MS for probing protein-ligand interactions, and develops computational approaches for predicting collision cross sections of structurally diverse molecules.Chapter 2 presents a systematic evaluation of separation parameters and calibration strategies for a high-resolution Traveling Wave-SLIM (TW-SLIM) ion mobility system. We systematically evaluate the impact of traveling wave (TW) parameters on analysis time, peak-to-peak resolution, resolving power, and root-mean-square deviations of calibration residuals for calibrant ions using a SLIM-based high-resolution ion mobility platform. We identify a regime at which ions travel at the wave velocity rather than being separated based on mobility as a unifying explanation for degradation in both separation quality and calibration accuracy. We also compare trinomial and shifted-exponential calibration functions and introduce a novel approach using the shifted-exponential function that reduces bias from limited precision in reference CCS values by approximately four-fold. Applying our novel approach and optimized parameters, we assess the precision of CCS values for small molecule drugs and demonstrate baseline separation of protonation-induced verapamil conformers, achieving highly precise relative CCS values, typically under 300 parts per million (ppm), marking significant progress in isomer-specific assays and molecular identification. Chapter 3 details the application of native mass spectrometry to characterize drug binding to human liver fatty acid binding protein 1 (hFABP1). This work, part of a collaborative study, demonstrates that drugs form ternary complexes with hFABP1 and endogenous lipids. Native MS served multiple critical roles: monitoring copurifying molecules during protein purification, confirming the binding stoichiometry of the fatty acid probe DAUDA with hFABP1, and providing unambiguous verification of ternary complex formation. Using native MS, we directly detected DAUDA-hFABP1-diclofenac ternary complexes, demonstrating that drugs can bind to hFABP1 without fully displacing endogenous lipids. These results have implications for understanding how FABP1 binding may alter drug metabolism and clearance in the liver. Chapter 4 systematically examines how ammonium acetate concentration affects electrokinetic nano-electrospray ionization and native MS performance across 2 mM to 1000 mM in both positive and negative polarities, using droplet size, flow rate measurements, and native mass spectra of intact proteins as diagnostics. Droplet size and flow rate depended strongly on concentration but were independent of polarity, while voltage–current relationships differed markedly between modes: negative mode operated under a narrower window, limiting accessible concentrations to below approximately 800 mM. In positive mode, the adverse effects of sodium chloride on spectral quality decreased monotonically with increasing ammonium acetate concentration, with continued improvement up to 1000 mM; in negative mode, this approach was substantially less effective, as the high concentrations required exceeded the stable operating window. These results establish that ionization polarity is a primary variable in native MS method development and that positive and negative mode nanoESI operate under fundamentally different constraints when nonvolatile salt contamination is present. Chapter 5 introduces MobileMesh, an open-source Python library that calculates collision cross sections using triangle mesh representations of electron density isosurfaces, eliminating the need for element-specific parameterization. Characterizing petroleum at the molecular level requires analytical techniques sensitive to both elemental composition and molecular structure, and existing computational methods for calculating CCS rely on element-specific parameters that must be recalibrated across different ion classes, limiting their accuracy for the structurally diverse and heteroatom-rich species found in petroleum. MobileMesh addresses this limitation using specular scattering algorithms adapted from ray casting techniques in computer graphics, leveraging the Open3D library for high-performance triangle mesh operations. MobileMesh was evaluated against experimental helium CCS for radical cations of polycyclic aromatic hydrocarbons, a class of ions particularly challenging for traditional methods due to their delocalized charge and open-shell electronic structure, yielding strong agreement with experimental values without any element-specific fitting. By relying exclusively on stable, open-source software, MobileMesh reduces computational requirements from tens of thousands of processor cores to seconds on a single desktop computer, making electron density isosurface-based CCS calculations broadly accessible to the ion mobility community for the first time. Overall, the methods and findings presented in this dissertation advance the quantitative rigor of ion mobility measurements, deepen our understanding of electrospray ionization processes, demonstrate the utility of native MS for probing protein-ligand interactions, and introduce computational approaches for predicting collision cross sections of structurally diverse molecules. Collectively, these contributions provide practical guidance and new tools that can be broadly adopted by the IM-MS community to enhance molecular characterization workflows.application/pdfen-USCC BY-NC-NDAnalytical ChemistryComputational CharacterizationIon MobilityMolecular CharacterizationNative Mass SpectrometryProtein CharacterizationChemistryBiophysicsChemistryThesis