Chemistry & Chemical Biology Department Theses and Dissertations

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Advances in Gas Chromatography and Vacuum UV Spectroscopy: Applications to Fire Debris Analysis & Drugs of Abuse
(2020-12) Roberson, Zackery Ray; Goodpaster, John V.; Manicke, Nicholas E.; Webb, Ian K.; Laulhé, Sébastien
In forensic chemistry, a quicker and more accurate analysis of a sample is always being pursued. Speedy analyses allow the analyst to provide quick turn-around times and potentially decrease back-logs that are known to be a problem in the field. Accurate analyses are paramount with the futures and lives of the accused potentially on the line. One of the most common methods of analysis in forensic chemistry laboratories is gas chromatography, chosen for the relative speed and efficiency afforded by this method. Two major routes were attempted to further improve on gas chromatography applications in forensic chemistry. The first route was to decrease separation times for analysis of ignitable liquid residues by using micro-bore wall coated open-tubular columns. Micro-bore columns are much shorter and have higher separation efficiencies than the standard columns used in forensic chemistry, allowing for faster analysis times while maintaining the expected peak separation. Typical separation times for fire debris samples are between thirty minutes and one hour, the micro-bore columns were able to achieve equivalent performance in three minutes. The reduction in analysis time was demonstrated by analysis of ignitable liquid residues from simulated fire debris exemplars. The second route looked at a relatively new detector for gas chromatography known as a vacuum ultraviolet (VUV) spectrophotometer. The VUV detector uses traditional UV and far-ultraviolet light to probe the pi and sigma bonds of the gas phase analytes as well as Rydberg traditions to produce spectra that are nearly unique to a compound. Thus far, the only spectra that were not discernable were from enantiomers, otherwise even diastereomers have been differentiated. The specificity attained with the VUV detector has achieved differentiation of compounds that mass spectrometry, the most common detection method for chromatography in forensic chemistry labs, has difficulty distinguishing. This specificity has been demonstrated herein by analyzing various classes of drugs of abuse and applicability to “real world” samples has been demonstrated by analysis of de-identified seized samples.
Advances in Gas Chromatography, Thermolysis, Mass Spectrometry, and Vacuum Ultraviolet Spectrometry
(2021-05) Rael, Ashur; Goodpaster, John V.; Manicke, Nicholas E.; Naumann, Christoph A.; Minto, Robert E.
In the area of forensic chemistry, improved or new analysis methods are continually being investigated. One common and powerful technique used in forensic chemistry is wall-coated open-tubular column (WCOT) gas chromatography with electron ionization single quadrupole mass spectrometry (GC-MS). Improvements to and effectiveness of alternatives to this instrumental platform were explored in an array of parallel inquiries. The areas studied included the column for the chromatographic separation, the universal detection method employed, and the fragmentation method used to enhance molecular identification. Superfine-micropacked capillary (SFµPC) columns may provide an alternative to commercial packed GC columns and WCOT GC columns that combines the benefits of the larger sample capacity of packed columns and the benefits of the excellent separation capabilities and mass spectrometry (MS) flow rate compatibility of WCOT columns. SFµPC columns suffer from high inlet pressure requirements and prior reported work has required specialized instrumentation for their use. Fabrication of and chromatography with SFµPC GC columns was successfully achieved with typical GC-MS instrumentation and within the flow rate limit of a MS. Additionally, the use of higher viscosity carrier gasses was demonstrated to reduce the required inlet pressure for SFµPC GC columns. Recently, a new vacuum ultraviolet spectrometer (VUV) universal detector has been commercialized for GC. The ability of VUV detectors to acquire absorbance spectra from 125 nm to 430 nm poses a potential alternative to MS. As such, GC-VUV provides an exciting potential alternative approach to achieving excellent quantitative and qualitative analysis across a wide range of analytes. The performance of VUV and MS detectors for forensic analysis in terms of quantitative and qualitative analysis was compared. Analysis of alkylbenzenes in ignitable liquids was explored, which can be important evidence from suspected arson fires and are difficult to differentiate with MS. The VUV detector was found to have superior specificity and comparable sensitivity to the MS detector in scan mode. Addition of thermolysis (Th) as an orthogonal fragmentation pathway provides the opportunity to increase the differences between MS fragmentation patterns. Fragmentation has been widely established to aid in identification of molecules with MS by providing characteristic fragments at characteristic relative abundances. However, molecules with very similar structures do not result in sizable spectral differences in all cases with typical MS fragmentation techniques. A series of Th units were fabricated and integrated into GC-Th-MS instruments. Th-MS was conducted with the thermally labile nitrate esters across a range of instrumentation and thermal conditions.
Advances in Solid Phase Microextraction for the Analysis of Volatile Compounds in Explosives, Tire Treatments, and Entomological Specimens
(2016-05) Kranz, William D.; Goodpaster, John V.; Manicke, Nick; Sardar, Rajesh; Picard, Christine Johanna; Long, Eric C.
Solid phase micro-extraction is a powerful and versatile technique, well-suited to the analysis of numerous samples of forensic interest. The exceptional sensitivity of the SPME platform, combined with its adaptability to traditional GC-MS systems and its ability to extract samples with minimal work-up, make it appropriate to applications in forensic laboratories. In a series of research projects, solid phase micro-extraction was employed for the analysis of explosives, commercial tire treatments, and entomological specimens. In the first project, the volatile organic compounds emanating from two brands of pseudo-explosive training aids for use in detector dog imprinting were determined by SPME-GC-MS, and the efficacy of these training materials was tested in live canine trials. In the second project, the headspace above various plasticizers was analyzed comparative to that of Composition C-4 in order to draw conclusions about the odor compound, 2- ethyl-1-hexnaol, with an eye toward the design of future training aids. In the third, automobile tires which had participated in professional race events were analyzed for the presence of illicit tire treatments, and in the fourth, a novel SPME-GC-MS method was developed for the analysis of blowfly (Diptera) liquid extracts. In the fifth and final project, the new method was put to the task of performing a chemotaxonomic analysis on pupa specimens, seeking to chemically characterize them according to their age, generation, and species.
Advancing the Applicability of Fast Photochemical Oxidation of Proteins to Complex Systems
(2016-08) Rinas, Aimee Lynn; Jones, Lisa; Georgiadis, Millie M.; Long, Eric C.; Manicke, Nicholas
Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced increases the complexity of identifying and quantifying oxidation products. The current methods available for quantifying the extent of oxidation either involve manual analysis steps, or limit the number of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. In addition to the data complexity, the peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. The recently published in cell FPOP method exemplifies where this field is headed - larger and more complex systems. This dissertation describes two new methodologies and one new technology for hydroxyl radical-mediated protein footprinting, expanding the applicability of the method. First is development of a new footprinting analysis method for both peptide and residue level analysis, allowing for faster quantification of results. This method utilizes a customized multilevel search workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in, expediting the analysis process. Second is the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Last is the design and implementation of a flow system for in cell FPOP, which hydrodynamically focuses the cells, and when used yielded a 13-fold increase in oxidized proteins and 2 orders of magnitude increase in the dynamic range of the method.
Analysis of TNT, DNA Methylation, and Hair Pigmentation via Gas Chromatography-Mass Spectrometry and Spectroscopic Techniques
(2019-08) Ruchti, Jacqueline; Goodpaster, John; Manicke, Nicholas; Picard, Christine
Base- and Visible Light-Promoted Activation of Aryl Halides under Transition-Metal-Free Conditions: Applications and Mechanistic Studies
(2021-12) Pan, Lei; Laulhé, Sébastien; Long, Eric; O'Donnell, Martin J.; Dai, Mingji
Aromatic rings are universal motifs in natural products, pharmaceuticals, agrochemicals, and wide variety of organic materials. Aromatic halides are widely used as synthetic precursors in all these applications. Therefore, tremendous effort has been devoted to activate aryl halides in the past decades. The common methods to activate aryl halides require the use of transition- metals either in the form of Grignard reagents or through the use of transition-metal catalysis. Over the past decade, photoredox catalysis has attracted significant attention as a cogent tool to develop greener synthetic processes and enable new molecular activation pathways under mild conditions. The most common of these approaches uses a photoredox/nickel dual catalytic cycle. While this technology has greatly expanded the toolbox of organic chemists, this method still requires expensive rare-metal-based catalyts. Herein, we present a series of visible light-induced methods that are transition-metal-free. These new base-promoted transformations and their mechanistic work will be discussed in the following order: We will first present our discovery that the dimsyl anion enables visible-light-promoted charge transfer in cross-coupling reactions of aryl halides. This work was applied to the synthesis of unsymmetrical diaryl chalcogenides. This method has a broad scope and functional group tolerance. An electron-donor-acceptor (EDA) complex between a dimsyl anion and the aryl halide is formed during the reaction and explains the observed aryl radical reactivity observed. Then, a visible-light-induced borylation and phosphorylation of aryl halides under mild conditions was developed. Inspired by the mechanistic breakthroughs observed in the previous work. The mechanism of this reaction also involves an aryl radical that is presumed to be formed also via an EDA complex. In other work, a photo-induced phosphonation of ArI using N,N- diisopropylethylamine (DIPEA) and trialkyl phosphites was developed. This method uses very mild conditions, which allowed the preparation a wide variety of functionalized aromatic phosphonates derivatives, including natural products and medicinal compounds. Finally, a photochemical amination of amides was developed via a C(sp 3 )–H bond functionalization process under visible light irradiation. This reaction showed good functional group compatibility without the use of external radical initiators, strong oxidants, or heat source. An EDA complex between N-bromophthalimide and LiOtBu is formed during the reaction.
Biochemical applications of DsRed-monomer utilizing fluorescence and metal-binding affinity
(2011-03-09) Goulding, Ann Marie; Deo, Sapna K.; Oh, Kyungsoo; Davidson, Amy; Simpson, Garth
The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.
Blood on FTA™ Paper: Does Punch Location Affect the Quality of a Forensic DNA Profile?
(2013-03-06) Carter, Megan Elizabeth; Picard, Christine; Siegel, Jay A.; Randall, Stephen Karl, 1953-
Forensic DNA profiling is widely used as an identification tool for associating an individual with evidence of a crime. Analysis of a DNA sample involves observation of data in the form of an electropherogram, and subsequently annotating a DNA “profile” from an individual or from the evidence. The profile obtained from the evidence can be compared to reference profiles deposited in a national DNA database, which may include the potential contributor. Following a match, a random match probability is calculated to determine how common that genotype is in the population. This is the probability of obtaining that same DNA profile by sampling from a pool of unrelated individuals. Each state has adopted various laws requiring suspects and/or offenders to submit a DNA sample for the national database (such as California’s law that all who are arrested must provide a DNA sample). These profiles can then be associated with past unsolved crimes, and remain in the database to be searched in the event of future crimes. In the case of database samples, a physical sample of the offender’s DNA must be kept on file in the laboratory indefinitely so that in the event of a database hit, the sample is able to be retested. Current methods are to collect a buccal swab or blood sample, and store the DNA extracts under strict preservation conditions, i.e. cold storage, typically -20° C. With continually increasing number of samples submitted, a burden is placed on crime labs to store these DNA extracts. A solution was required to help control the costs of properly storing the samples. FTA™ paper was created to fulfill the need for inexpensive, low maintenance, long term storage of biological samples, which makes it ideal for use with convicted offender DNA samples. FTA™ paper is a commercially produced, chemically treated paper that allows DNA to be stored at room temperature for years with no costly storage facilities or conditions. Once a sample is required for DNA testing, a small disc is removed and is to be used directly in a PCR reaction. A high quality profile is important for comparing suspect profiles to unknown or database profiles. A single difference between a suspect and evidentiary sample can lead to exclusion. Unfortunately, the DNA profile results yielded from the direct addition have been unfavorable. Thus, most crime laboratories will extract the DNA from the disc, leading to additional time and cost to analyze a reference sample. Many of the profiles from the direct addition of an FTA™ disc result in poor quality profiles, likely due to an increase in PCR inhibitors and high concentrations of DNA. Currently, standardized protocols regarding the recommended locations for removal of a sample disc from a bloodspot on an FTA™ card does not exist. This study aims to validate the optimal location by comparing DNA profiles obtained from discs removed from the center, halfway, and edge locations of a bloodspot from 50 anonymous donors. Optimal punch location was first scored on the number of failed, partial or discordant profiles. Then, profile quality was determined based on peak characteristics of the resulting DNA profiles. The results for all three disc locations were 5.3% failed amplifications, 4.2% partial amplifications, and one case of a discordant profile. Profile quality for the majority of the samples showed a high incidence of stutter and the absence of non-template adenylation. Of the three disc locations, the edge of the blood stain was ideal, due to a presumably lower concentration of DNA and likely more dilute amount of the PCR inhibitor heme. Therefore, based on the results of this study, there is a greater probability of success using a sample from the edge of a blood stain spotted in FTA™ paper than any other location of the FTA™ card.
Characterization of a fatty acid elongase condensing enzyme by site-directed mutagenesis and biochemical analysis
(2014) Hernandez-Buquer, Selene; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Li, Lei
Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.
Chemometric Analysis of Volatile Organic Compound Biomarkers of Disease and Development of Solid Phase Microextraction Fibers to Evaluate Gas Sensing Layers
(2022-08) Woollam, Mark David; Agarwal, Mangilal; Deiss, Frédérique; Goodpaster, John; Naumann, Christoph
Canines can detect different diseases simply by smelling different biological sample types, including urine, breath and sweat. This has led researchers to try and discovery unique volatile organic compound (VOC) biomarkers. The power of VOC biomarkers lies in the fact that one day they may be able to be utilized for noninvasive, rapid and accurate diagnostics at a point of care using miniaturized biosensors. However, the identity of the specific VOC biomarkers must be demonstrated before designing and fabricating sensing systems. Through an extensive series of experiments, VOCs in urine are profiled by solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) to identify biomarkers for breast cancer using murine models. The results from these experiments indicated that unique classes of urinary VOCs, primarily terpene/terpenoids and carbonyls, are potential biomarkers of breast cancer. Through implementing chemometric approaches, unique panels of VOCs were identified for breast cancer detection, identifying tumor location, determining the efficacy of dopaminergic antitumor treatments, and tracking cancer progression. Other diseases, including COVID-19 and hypoglycemia (low blood sugar) were also probed to identify volatile biomarkers present in breath samples. VOC biomarker identification is an important step toward developing portable gas sensors, but another hurdle that exists is that current sensors lack selectivity toward specific VOCs of interest. Furthermore, testing sensors for sensitivity and selectivity is an extensive process as VOCs must be tested individually because the sensors do not have modes of chromatographic separation or compound identification. Another set of experiments is presented to demonstrate that SPME fibers can be coated with materials, used to extract standard solutions of VOCs, and analyzed by GC-MS to determine the performance of various gas sensing layers. In the first of these experiments, polyetherimide (PEI) was coated onto a SPME fiber and compared to commercial polyacrylate (PAA) fibers. The second experiment tuned the extraction efficiency of polyvinylidene fluoride (PVDF) - carbon black (CB) composites and showed that they had higher sensitivity for urinary VOC extraction relative to a polydimethylsiloxane (PDMS) SPME fiber. These results demonstrate SPME GC-MS can rapidly characterize and tune the VOC adsorption capabilities of gas sensing layers.

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