Improvements in printing technology and the wide accessibility of advanced printers has resulted in an increase in counterfeiting. Of particular interest to forensic document examiners, ink analysis of security documents has emerged as an important tool for the analysis, comparison, and association of inks to a potential production source. In this study, methods were developed for Py-GC/MS, ATR-FTIR, DART-MS, and MALDI-MS analyses of printing inks of four classes: 78 inkjet inks, 76 toners, 79 offset inks, and 86 intaglio inks in order to generate information on the organic chemical characterization of the inks to determine the ability to associate and discriminate the inks for uses in security document examinations. The suite of analytical techniques evaluated in the study focused on having the following analytical characteristics: 1) rapid analysis time, 2) little-to-no sample preparation, 3) minimal destructiveness to the sample, 4) allow for association of inks with the same source of origin as well as discrimination of inks originating from different sources. As a result of this study, a novel searchable library database of inks was developed for use with each analytical chemical method that allows for data fusion. Py-GC/MS analysis was used to achieve >63% discrimination of toner inks on the basis of the characterization of polymer degradation products. The combination of a protocol implementing first ATR-FTIR and subsequently DART-MS analysis resulted in >96% discrimination for toners, 95% for inkjet, >92% for offset, and >54% for intaglio. In addition, a case study was performed using DART-MS to illustrate its utility as a tool for ink analysis in document examination. The results of MALDI-MS analyses from two different instruments resulted in >90% discrimination of a subset of all inks by characterizing the colorant molecules present in ink formulations. A study characterizing luminescent compounds present in the formulation of inks in crossed-line intersections was performed using a combination of MALDI-MS, LC-MS, and TLC. Overall, it was shown that the combination of analytical techniques included within this dissertation can provide information on the organic chemical composition of four classes of printing inks, which is useful for the future of document examination.

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This dissertation presents a comprehensive study of the peptides of interest to deeper understand the gas-phase acid-base properties in relation to their conformations and chirality. In the first part of the study, two pairs of alanine (A)-based isomeric peptides consisting of a basic probe, lysine (Lys) or 2,3-diaminopropionic acid (Dap), were investigated to understand the nature of the enhanced basicity when the basic probe was moved from the N-terminus to the C-terminus. In the second part of the study, alanine-based peptides containing a cysteine (C) as the acidic probe were investigated to understand the chirality effects on the gas-phase acidity by altering the chiral centers systemically. Previous studies by mass spectrometry showed that the peptides ALys and AADap have had remarkably higher proton affinity (PA) compared to their isomeric counterparts LysA and DapAA. In this work, conformations, energetics, and molecular properties of the peptide systems have been thoroughly characterized through infrared multiple photon dissociation (IRMPD) spectroscopy and quantum chemical computations utilizing a set of molecular modeling tools. The molecular properties include charge distribution, dipole moment, torsional strain, hydrogen bonding, and non-covalent interaction. Computational studies yielded the lowest energy conformations along with their theoretical infrared (IR) spectra for each of the peptide systems. The resulting theoretical proton affinities are in excellent agreement with experiments. The results also suggest that the relative stability of the protonated peptides is the main source of the difference in the gas-phase basicity between the isomeric peptides. Structurally representative conformations for the protonated peptides were identified by matching the theoretical IR spectra to the corresponding IRMPD spectra. The band features of the IRMPD spectra were analyzed in detail by vibrational mode decomposition. The N-probe peptide ions, LysAH+ and DapAAH+, adopt diverse backbone geometries and intramolecular hydrogen bonding networks, and rely heavily on the hydrogen bonds for conformational stabilization. In contrast, the C-probe peptide ions, ALysH+ and AADapH+, adopt helical conformations, and benefit from the interaction between the helix macrodipole and the charged NH3+ group. The low torsional strain on the Lys sidechain contributes significantly to the conformational stability for ALysH+ than for LysAH+. The chirality of each residue in CAAA and Ac-CAAA (Ac represents the acetyl group) alters from the L- to the D-form systematically to generate two series of peptides. Qualitative comparison of the gas-phase acidity was achieved through mass spectrometry measurements using the Cooks’ kinetic method. The following two acidity ladders from the most acidic to the least acidic were obtained: CAAdA > CAdAA ~ CAAA > dCAAA > CdAAA, and Ac-dCAAA > Ac-CAAdA > Ac-CAAA > Ac-CAdAA > Ac-CdAAA, where the superscript-d in front of the amino acid symbol indicates the D-form of that residue. In both non-acetylated and acetylated peptides, the gas-phase acidity increases as the D-alanine moves further away from the N-terminal acidic probe cysteine. Inversion of the cysteine residue from the L- to the D-form reduces the gas-phase acidity of the non-acetylated peptide and enhances the gas-phase acidity of the acetylated one. Overall, the change in the gas-phase acidity is likely due to the conformational reorganization in the deprotonated peptides upon chiral inversion.

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<p>The human interactome presents a goldmine of potentially powerful therapeutic targets, yet very few small molecule modulators of protein-protein interactions (PPI) have been identified. PPI pose a particular challenge for drug discovery, and one of the major obstacles to fully exploiting these interactions is a lack of appropriate technologies to screen for modulating compounds. This thesis aims to address the need for function- based approaches that target PPI by using magnetic beads (MB) and mass spectrometry (MS) to develop efficient assays to monitor these interactions and their modulation by small molecules. The work begins with the validation of a novel magnetic “fishing” assay, which uses affinity-capture MB to isolate intact complexes of a “bait” protein from solution. By monitoring the recovery of the secondary binding partner, this assay was used to functionally screen a library of 1000 compounds for small molecule modulators of a calmodulin/melittin (CaM/Mel) model system. The versatility of magnetic “fishing” is clearly demonstrated during a study of a more relevant CaM-based system, which uncovered a novel mode of interaction for the CaM-binding domain of transcription factor SOX9. In addition to the MB-based approach, a simple MS-based competitive displacement assay is developed to identify minimal inhibitory fragments of a target complex as indicators of potential ‘hot-spots’. The assay was used to probe a DNA repair complex of XRCC4/ligaseIV, and identified a short helix that can be used as a more defined target surface for future high-throughput screening and rational drug design. The functional MS-based assays herein are highly adaptable tools to monitor PPI, and will facilitate the study of these and other important biomolecular interactions.</p> / Doctor of Philosophy (PhD)

<p>The comprehensive analysis of intact lipids (called lipidomics) can provide information about the presence of microbial communities in an ecosystem and assist in understanding the biogeochemistry in that system. In previous work we had developed a method to determine the profiles of eight phospholipid classes in a soil microorganism by direct-infusion electrospray mass spectroscopy using tandem mass spectrometry. The work done in this study encompasses first the optimization of previous methodology for use with water and sediment samples containing low concentration of phospholipids and large amounts of organic contaminants and secondly the application of this method to the analysis of phospholipids within composite tailings and recycled process water using a triple quadrupole mass spectrometer to determine the intact lipids in the bacterial community. The results are presented illustrating the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids present in composite tailing samples and recycled process water. This thesis begins with the optimization of a direct infusion mass spectrometry method, which allowed the analysis of intact phospholipids within both water and sediment samples. This method allows for high through-put analysis using both the separation afforded by neutral loss and precursor ion scanning modes and a database containing all possible adduct masses to identify and quantify unknown phospholipids. This method was then applied to water and sediment samples obtained from the Syncrude Sandhill Fen composite tailings site. This analysis discovered multiple differences within the water samples attributed to changes both in well temperature and the ongoing reclamation projects resulting in the change in phospholipid profiles. This thesis also outlines the shortcomings of the direct infusion lipidomics method when used for the analysis of complex samples such as composite tailings sediment samples. In summary, this thesis has demonstrated that direct infusion lipidomics can be successfully applied to the analysis of water samples and yield statistically significant differences within the microbial lipidome.</p> / Master of Science (MSc)

<p>Research in this thesis has focused on development and application of novel methodologies that enhance sample throughput and data fidelity when performing untargeted metabolome profiling by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Metabolomics is a valuable tool in functional genomics research to investigate underlying molecular mechanisms associated with human health since metabolites are “real-world” end-products of gene expression. CE-MS is well-suited for metabolomics because it is a high efficiency microseparation technique that can be used to resolve complex mixtures of polar metabolites in human biofluids without complicated sample workup. In this thesis, a novel CE-MS assay for estrogens and their intact ionic conjugates has been described (<em>Chapter II</em>) to expand metabolome coverage that enables resolution of positional isomers with high selectivity. This is critical for better understanding of underlying perturbations in estrogen metabolism since the biological activity of estrogens are dependent on specific primary and secondary metabolic transformations. MSI-CE-MS has been introduced as a high-throughput approach for large-scale metabolomic studies based on serial injection of multiple segments of sample within a single fused-silica capillary (<em>Chapter III</em>). It reduces analysis times while increasing data quality and confidence in peak assignment together with better quality assurance. An accelerated workflow for metabolomics has also been developed when using MSI-CE-MS, where a dilution trend filter is used as a primary screen to authenticate reproducible sample-derived metabolites from a pooled sample while eliminating spurious artifact and background signals. In this way, complicated time alignment and peak picking algorithms are avoided when processing data in metabolomics to reduce false discoveries. This strategy was subsequently used in two metabolomics applications (<em>Chapters IV</em> and <em>V</em>) to identify plasma markers associated with strenuous exercise and adaptive training responses following a six-week high intensity interval training. The impact of exercise intervention to improve the glucose tolerance of a cohort of overweight/obese yet non-diabetic women was investigated on an individual level when using a cross-over design. Personalized interventions are critical in designing more effective therapies to prevent metabolic diseases due to inter-subject variations in treatment responses, including potential adverse effects. MSI-CE-MS offers a revolutionary approach for biomarker discovery in metabolomics with high sample throughput and high data fidelity, which is critical for validation of safe yet effective lifestyle interventions that promote human health and reduce risk for chronic diseases.</p> / Doctor of Philosophy (PhD)

<p>Fabrication of microarrays using sol-gel immobilization has been utilized as an approach to develop high density biosensors. Microarray fabrication using various printing techniques including pin-printing and piezoelectric ink jet printing methods has been demonstrated. However, only limited characterization to understand the encapsulated biomolecule-material interface has been reported. Herein, Chemical characterization using X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR) on pin-printed microarrays of sol-gel derived acetylcholinesterase (AChE) microarrays is reported. Furthermore, the <em>in situ</em> fabrication of microarrays following the sol-gel process using piezoelectric ink jet printing methods was explored. Through techniques measuring solution viscosity, surface tension and particle size, important aspects of bio-ink formulation for piezoelectric ink jet printing were identified. Combined, a greater understanding towards the fabrication and characterization of sol-gel derived microarrays was achieved through this exploratory research.</p> / Master of Science (MSc)