The role of solvent-analyte interactions in determining ionization probability in laser electrospray mass spectrometry (LEMS)

In chemical and biomedical research, one of the most important goals remains as to create advanced analytical methods that can analyze biomolecules in a sensitive and selective way. When used on difficult systems such as hydrophobic peptides, membrane proteins, or spatially heterogeneous tissues, traditional methods, like electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), face many challenges. Some of these challenges are, poor ionization efficiency in the presence of salts or detergents, destabilization of conformations during analyzing, and the requirement for matrix application or extensive sample preparation. This dissertation investigates the capability of Laser Electrospray Mass Spectrometry (LEMS) in addressing those challenges. Specifically in the areas of chemical analysis, method development, and molecular imaging. LEMS uses femtosecond laser ablation and electrospray ionization together to detect intact biomolecules from complex environments. Additionally, LEMS offers ambient and label-free analysis. The main goal of this work is to improve our basic understanding of LEMS through a series of studies that optimize the instrumental components, validate performance, and show the applicability of LEMS to biologically relevant systems. The second chapter focuses on the use of LEMS to study membrane-associated peptides, using gramicidin as a model hydrophobic system. Membrane proteins are important in cellular signaling, ion transport, and drug targeting. But these proteins are not well characterized in proteomic studies. This is because of their amphipathic nature and poor solubility in aqueous environments. LEMS analysis was conducted on gramicidin in organic solvents, detergents, and liposomes to evaluate how the surrounding environment effect ionization and structural stability of gramicidin. High-quality mass spectra were obtained in methanol and non-ionic detergents such as DDM, while ionic detergents such as SDS disrupted native peptide folding by specific interactions with tryptophan residues. Incorporation into DPPC liposomes provided a more biologically relevant bilayer environment. Incorporation of gramicidin into liposome bilayer was confirmed via differential scanning calorimetry. The study showed that the solvent polarity, detergent charge, and sample preparation technique (sonication vs. extrusion) all played important roles in determining peptide solubilization and LEMS signal quality. Ion binding analysis showed that gramicidin forms complexes with both monovalent and divalent metal cations. Nonlinear modeling of binding data also showed that ion selectivity patterns depend on the solvent used in the experiments. This chapter shows that LEMS is a sensitive way to study membrane-active peptides in a wide range of physical and chemical settings. Chapter three explores the application of LEMS in mass spectrometry imaging (MSI) for the spatial analysis of biochemical distributions in tissue. First set of imaging experiments were on studying of mouse brain sections following traumatic brain injury (TBI). TBI is a condition involving complex metabolic and lipidomic changes in the brain. LEMS-MSI experiments showed that the injury caused molecular changes including lysophosphatidylcholine (LPC) and heme b, and hemoglobin subunits that were localized to hemorrhagic regions. Principal component analysis (PCA) was used to determine important features in the high-dimensional MSI dataset. PCA was able to identify spatially resolved signals for adenosine, uridine, and other potential biomarkers. Imaging experiments were further conducted by evaluating laser pulse duration (femtosecond vs. picosecond vs. nanosecond) and electrospray emitter diameter. Femtosecond pulses provided the optimum ablation with minimal thermal damage. Nano-ESI emitter gave the most stable and sensitive signal among the emitters that were tested. These results show that LEMS-MSI is a label-free method for tissue analysis that preserves spatial detail and biochemical specificity. LEMS also avoid artifacts associated with matrix deposition or sample dehydration. The fourth chapter investigates how the dielectric constant of the solvent used in sample preparation affects the formation of ions, the distribution of charge states, and the aggregation of peptides in LEMS. A series of binary solvent mixtures with varying dielectric constants was prepared by mixing methanol with water, ethanol, propanol, or butanol. Mass spectra of gramicidin A showed that high-dielectric solvents favored protonated ions and doubly charged species. Conversely, low-dielectric solvents favored sodium adduction and singly charged ions. Thermodynamic data on ion hydration and electrostatic stabilization were used to explain these trends. Further experiments analyzed ionization of three gramicidin isoforms (A, B, and C), which differ at a single amino acid residue. Results showed that selectivity depend on polarity. For example, gramicidin C (with a hydrophilic tyrosine residue) showed increased ionization in polar solvents. In contrast, gramicidin B (with a phenylalanine) showed a decreased mass spectrometry signal, consistent with the hydrophobicity and poor solvation of gramicidin B in polar solvents. The polarity of solvent also affected the formation of non-covalent dimers. For example, gramicidin A dimers were stabilized in hydrophobic solvents and disrupted in aqueous mixtures. These findings provide more insight into how the solvent environment affects analyte ionization and conformation in LEMS. compared to ESI, where all species are co-solvated. Overall, the research described in this dissertation shows that LEMS can be strategically improved and applied to a wide range of analytical problems involving peptides, proteins, and tissue. This work defines the experimental conditions that yield high sensitivity, structural preservation, and spatial resolution by careful evaluation of solvents, sample matrices, electrospray configurations, and laser parameters. In this work, LEMS is shown to tolerate complex sample environments and also distinguish closely related biomolecules based on polarity. Additionally, LEMS-MSI reveal biologically meaningful chemical distributions in tissue. These features make LEMS a powerful alternative to traditional mass spectrometry techniques for method development, biomarker discovery, and the study of membrane active peptides under native like conditions.