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Browsing by Subject "Mass spectrometry"

Now showing 1 - 13 of 13
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    Automated quantification of 13C labeled peptides
    (2013-08) Goldford, Joshua Elliot
    Metabolic flux analysis (MFA) is a technique used to elucidate intracellular reaction rates (fluxes) in a metabolic network. Intracellular fluxes are determined by providing substrate enriched with stable, heavy isotopic label and subsequently measuring the incorporation of label into metabolic end products. This results in metabolic end products consisting of isomers of discrete mass states, termed isotopomers. The resulting isotopomer distributions (MIDs) for each metabolic end product are then used to infer fluxes. Typically, metabolic end products used for MFA are derivatized protein-bound amino acids. Protein is extracted from the sample and hydrolyzed into constitutive amino acids, resulting in a amino acid pools derived from all cellular protein. Each amino acid pool contains amino acids potentially synthesized from different subcellular compartments, subspecies within a culture, or from different time points within the cell cycle. Thus, fluxes inferred from hydrolyzed total protein lack spatial and temporal resolution. However, if amino acid MIDs were to be measured directly from individual proteins, one could derive the fluxes at the time and place for which that particular protein was synthesized. Therefore, obtaining amino acid MIDs from individual proteins could enable spatial and temporal resolution for metabolic flux analysis. One solution would be to purify individual protein and hydrolyze and measure amino acid MIDs. This approach would require a significant amount of protein, is manually intensive and expensive. A much more viable solution utilizes high-throughput and high-resolution mass spectrometry to quantify and identify peptide MIDs, which can be used to infer constitutive amino acid MIDs. However, there is no well-defined, automated framework for the extraction and quantification of peptide MIDs from raw mass spectra.In the first chapter, the conceptual framework and vocabulary need for mass spectrometry and peptide-based MFA are provided, with a statistical emphasis. Chapter 2 provides a review of proteomics instrumentation for peptide based MFA followed by the algorithmic considerations and potential software solutions available for the extraction of peptide MIDs.Chapter 3 will describe the methods developed for the automated extraction and quantification of isotopically enriched peptides, including parameter optimization of existing methods and description of novel clustering and quantification methods. Chapter 4 describes the validation of the methods using three different sets of labeled peptide MIDs. Chapter 5 provides a brief discussion of method and software improvements for both identification and quantification followed by a brief discussion of future work.
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    Biomonitoring Exposures to Environmental and Dietary Carcinogens by Targeted and Untargeted Mass Spectrometry
    (2022-07) Konorev, Dmitri
    Humans are exposed to a wide variety of exogenous chemicals that may be implicated in DNA damage and cancer. Typical sources of carcinogen exposure include the diet, environment, and tobacco smoke. There is an epidemiological link between smoking cancer, as well as cooked and red meat consumption, and cancer. Despite extensive study, the chemicals responsible for carcinogenesis are unconfirmed. Specific and sensitive markers of DNA damage by discrete chemicals are needed confirm existing paradigms of carcinogenesis. Chapter 1 outlines the epidemiology and mechanisms by which chemicals from the diet and tobacco can lead to colorectal cancer and bladder cancer. Tobacco smoking is a well-established cause of bladder cancer, as is occupational exposure to high levels of carcinogenic aromatic amines, such as 4-aminobiphenyl and 2-naphthylamine, which are also present in tobacco smoke at low levels. The levels of these compounds in tobacco smoke may be insufficient to bladder carcinogenesis. Other related compounds, such as alkylaniline derivatives, and structurally-related heterocyclic aromatic amines, are present at much higher levels in tobacco smoke and may be risk factors for bladder cancer. Chapter 2 outlines our methodologies to assay a nonpolar, basic fraction of tobacco smoke condensate, containing aromatic amines and heterocyclic aromatic amines, by liquid chromatography coupled to mass spectrometry (LC/MS). These compounds were measured by targeted and untargeted LC/MS as well as with the use of high field asymmetric waveform ion mobility spectrometry coupled to LC/MS to add an additional dimension of separation and reduce sample complexity. Chapter 3 presents our original research in the development of highly sensitive, validated LC/MS methods to measure DNA adducts and abasic sites from cooked meat and tobacco carcinogens in colorectal tissue. These methods were developed using a rat animal model dosed with carcinogens that form DNA adducts associated with colorectal cancer based on previous epidemiological and mechanistic evidence. In Chapter 4, we applied this methodology to human colorectal DNA samples from colorectal cancer patient biopsy samples. We did not detect DNA adducts of exogenous carcinogens but did detect endogenously-formed DNA and abasic sites in these samples. We then contextualized these results within greater paradigms of colorectal cancer carcinogenesis.
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    Covalent Reactions Between Flavors and the Model Protein β-Lactoglobulin
    (2020-07) Anantharamkrishnan, Vaidhyanathan
    Demand for high protein plant and dairy-based diets has been increasing but delivering them has become problematic for the food industry. The flavor issues are due to multifaceted interactions that occur between food proteins and flavoring components. Over 40 years of research has been done on studying temporary interactions between flavor and proteins, but very little work has been done on more permanent interactions – covalent bonding. Covalent bonding takes place between the side chains and terminal amino acids of food proteins and reactive aroma compounds that will change the flavor profile of the product in a permanent manner. β- lactoglobulin (BLG) was chosen as a model protein for this study as it is well characterized in both amino acid sequence and structure, its molecular weight is suitable for intact protein mass spectrometry and it is a major protein used in food industry. This thesis study developed a methodology using UPLC-ESI/qTOF-MS for monitoring the nature and extent of the covalent reactions based upon the change in molecular weight (Protein + flavor) that occurs after reaction. The cross linking of protein with flavor compounds was evaluated using gel electrophoresis. A proteomics approach using LC and tandem MS after enzymatic digestion was taken to identify the sites of post-translational modification between the flavor compounds and the BLG protein. The UPLC-ESI/qTOF-MS methodology in tandem with proteomics and gel electrophoresis yield a detailed view of flavor/BLG interactions that offered insights on minimizing these undesirable reactions in the future. A flavoring typically is created from a mixture of volatile chemicals that generally comprise a variety of functional groups. Some of these flavor components when added to a protein matrix form covalent adducts resulting in a change in flavor character or a loss of its potency. The end result of these reactions create an imbalanced flavor, one that is not acceptable to a consumer. This research study analyzed 47 different flavor compounds from 13 different functional groups for their covalent adduct formation with BLG. Aldehydes, sulfur-containing molecules (especially thiols), and functional group-containing furans were found to be the most reactive of the flavor components studied. Thiol-containing compounds reduced disulfide linkages in BLG to result in disulfide interchange and formation of new disulfide linkages with the free cysteine group. Ketones were generally stable, but α-diketones (e.g., diacetyl) were reactive. Some bases (e.g., pyrazines and pyridines) were non-reactive, while the nucleophilic allylamine was reactive. Hydrocarbons, alcohols, acids, esters, lactones, and pyrans did not give observable levels of adduct formation within the time period studied. Due to the varied environmental conditions present in various food systems, the nature and extent of covalent interactions would likely change. This study investigated the influence of pH, temperature and water activity on the covalent adduct formation between BLG and selected flavor molecules. Covalent adduct formation was slower in acidic pHs. The rate and extent of the reaction increased with increasing pH. The rate of formation of adducts increased with temperature. Higher temperatures (45°C) caused the formation of products that were not observed at lower temperatures (4°C and 25°C). An increase in water activity lead to an increase in formation of adducts for allyl isothiocyanate. There were no observable differences for the effect of water activity on the reaction rate for benzaldehyde, citral and dimethyl disulfide. Results will help in understanding the conditions at which flavor compounds will covalently bond with a protein and ways to potentially avoid it. Thereby, helping the food industry to develop flavor protein matrices that have a longer shelf life.
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    Fundamentals of a systems biology approach to In Vitro tissue growth
    (2013-05) Beck, Richard Joseph
    Tissue engineering needs a paradigm shift in order to generate clinically useful products. The field has yet to regularly produce implantable tissue-engineered products. The conventional manner in which input stimuli are applied without consideration of current cellular activity level is certainly suboptimal. The objective of this line of research is to produce a method for rationally choosing input stimuli that drive the cells toward optimal tissue growth. Transient phosphorylation of signaling proteins after a perturbation in stimuli contains biological information concerning downstream tissue growth. The overall project aims to build a statistical model predictive of tissue growth via information of the upstream phosphoproteome minutes after a change in stimuli. The validity of such a statistical model can be tested based on its utility to direct tissue growth: stimuli will be chosen on the basis of which corresponding phosphoproteome profile(s) is predicted to yield the best downstream tissue growth; this can be directly compared to conventional tissue engineering methods. This doctoral project focused on obtaining sample types and tailoring methods appropriate for a systems biology and statistical approach, especially in regard to the label-free quantification of phosphopeptide enrichments. Neonatal human dermal fibroblasts (nhDF) were expanded to near confluence, at which point basal medium for tissue production was applied. After two days, nhDF were perturbed with basal medium supplemented with 1 or 10 ng/mL TGF-β1. Cells were harvested at 10, 20, or 30 minutes for intracellular proteins. Resultant protein lysates were digested to peptides via trypsin and enriched for phosphopeptides via Iron Immobilized Metal Affinity Chromatography (IMAC). Phosphopeptide enrichments were analyzed by tandem mass spectrometry. A total of 1689 peptides were both identified with phosphorylation and quantified using distinct algorithms. Under strict statistical tests, 22 of these peptides were found to differ between treatments/time. Corresponding downstream collagen deposition was also found to differ between treatments. These results indicate that the type of quantitative data needed for the overall project can be acquired. The methods developed can be used in finding a statistical relationship between tissue growth and upstream phosphoproteome profiles.
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    High performance liquid chromatography with tandem laser induced fluorescence and mass spectrometry detection methods to monitor the metabolic profile of doxorubicin and its alteration with age
    (2010-09) Katzenmeyer, Joseph Bradly
    In this thesis, a combination of high-performance liquid chromatography (HPLC), laser-induced fluorescence (LIF) and mass spectrometry (MS) is used to assess the metabolic profile of doxorubicin in vitro using the post-mitochondrial fraction (PMF) from the liver of Fischer 344 rats. Monitoring the metabolism of xenobiotics and drugs is important in the development, screening and assessment of new chemical compounds for use in therapies. This often requires the use of two or more techniques in order to collect the relevant data. This is the case for the analysis of metabolic products of the anticancer drug doxorubicin. An HPLC instrument with tandem LIF and MS detection was developed and then used to quantify and identify the metabolic products of doxorubicin in vitro. Using this instrumentation, the consumption of doxorubicin and the appearance of 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone were monitored in rat liver post-mitochondrial fractions. This application demonstrates the potential of the tandem LIF-MS detection scheme in quantification and characterization of biotransformations of fluorescent xenobiotics of biomedical and environmental relevance. The HPLC-LIF-MS instrumentation and in vitro methods were then applied to investigate the changes in metabolism between young adult (10 months-old, 100% survival rate) and old (26 months-old, ~25% survival rate) Fischer 344 rats. Results suggest that with aging there is decrease in the rate of biotransformation of doxorubicin and that the timeframes needed to reach steady metabolite and doxorubicin levels are longer. On the other hand, the levels of metabolites and doxorubicin concentrations are not statistically different between the two age groups. In the future, the new methodologies presented here could be applied to investigate age-related changes in metabolism of drugs already in use, new compounds and xenobiotics with health relevance (e.g. pesticides or environmental pollutants).
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    Identifying candidate salivary oral cancer biomarkers:accurate protein quantification and analysis on LTQ type mass spectrometers.
    (2011-05) Onsongo, Getiria Innocent
    Cancer is one of the leading causes of death worldwide accounting for around 13 % of all deaths. Oral cancer in one of the more common cancers occurring more frequently than leukemia, brain, stomach, or ovarian cancer. Unfortunately, the 5-year survival rate for oral cancer has not significantly improved in the past 30 years and remains at approximately 50 %, in part, due to lack of reliable diagnostic biomarkers for early detection. It is estimated, if diagnosed and treated early, survival rates for oral cancer would significantly improve to between 80 % and 90 %. We need reliable reliable biomarkers for diagnosis and early detection of oral cancer. Recent developments in high-throughput proteomics techniques have made it possible to detect and identify low abundance proteins in complex biological fluids such as saliva. These low-abundance proteins could be a source of the elusive reliable biomarkers needed to improve survival rates for oral cancer. Limiting the widespread use of these proteomics techniques is lack of an accurate protein relative quantification technique. A typical high-throughput experiment identifies several thousand proteins with several hundred differentially abundant proteins. The cost of validating candidate biomarkers prevents validation of each differentially abundant protein to identify promising candidate biomarker. We need computational techniques to identify promising candidate biomarkers. This two-part dissertation presents: 1) a new technique for accurate protein relative quantification implemented in freely-available, open-source software (LTQ-iQuant) and 2) relational database operators for analyzing differentially abundant proteins to identify promising candidate biomarkers. Linear ion trap mass spectrometers, such as the hybrid LTQ-Orbitrap, are a popular choice for isobaric-tags based shotgun proteomics because of their advantages in analyzing complex biological samples. Coupled with orthogonal fractionation techniques, they can be used to detect low abundance proteins extending the range for detecting possible biomarkers. Limiting the widespread use of this combination for quantitative proteomics studies is lack of a technique tailored to LTQ type instruments that accurately reports protein abundance ratios, and is implemented in an automated software pipeline. This thesis presents a new technique implemented in a freely-available, open source software that fulfills this need. A major limitation of existing computational techniques when using high-throughput techniques is results that are too broad to be practically useful. A lot of the `potential' disease-specific biomarkers discovered have been found not to be specific to the disease being studied. They either belong to biological categories that change in response to infection or tissue injury, or are proteins whose changes are induced by other stresses such as medication and diet. This thesis extends the relational database engine to enable use of biological pathways to identify promising candidate biomarkers. Using biological pathways to analyze high-throughput data avoids results that are too broad to be practically useful. Protein differential abundance often is the criteria used to identify candidate biomarkers in high-throughput discovery-based biomarker studies. However, protein quantity by itself might not be the salient marker parameter. Protein function is often dependent on post-translational modifications such as phosphorylation and gylcosylation. By only using differential abundance to identify candidate biomarkers, we are limiting our ability to identify reliable biomarkers. We further develop new operators that in addition to using user specified pathways, use post-translational modification information to analyze high-throughput data. For the first time, we demonstrate feasibility of using post-translational modifications with relational database operators to analyze high-throughput proteomics data. Collectively, this work will facilitate the search for reliable biomarkers. LTQ-iQuant will make LTQ instruments and isobaric peptide tagging accessible to more proteomics researchers providing a new window into complex biological fluids. Relation operators will provide a systematic way of bridging the gap between unbiased data driven approach and hypothesis driven approach to prioritize candidate biomarkers.
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    Improving the detection of carbonylated peptides by mass spectrometry via solid-phase hydrazide enrichment and selective labeling with Oxygen-18 (18O)
    (2010-01) Roe, Mikel Robert
    Protein carbonylation is a post-translational oxidative protein modification known to alter protein function and impair cellular mechanisms. It is a relatively complex modification, characterized by a variety of structurally distinct reactive carbonyls that target a number of amino acid residues and originate via several different oxidative mechanisms. While identification of specific carbonylated proteins by mass spectrometry has provided insight regarding the protein pathways and complexes affected, the specific sites of carbonyl modification, necessary for determining the oxidative mechanisms involved as well as for explaining any associated functional consequences, are not routinely identified due to the relatively low abundance of carbonylated proteins. To address this issue, a number of methods for enriching carbonylated peptides have been developed, all of which involve derivatization with bulky reagents that often complicate the identification of peptides by tandem mass spectrometry. As an alternative to these label-based approaches, I have developed a label-free method for enriching carbonylated peptides that is based on their selective capture and controlled release from a novel solid-phase hydrazide reagent (SPH). The value of the SPH reagent method is demonstrated using a yeast lysate treated with the reactive lipid carbonyl 4-hydroxynonenal (HNE), where the use of pulsed-Q-dissociation (PQD) and neutral-loss triggered MS/MS/MS was employed for the first time to assist the identification of HNE-modified peptides by mass spectrometry. To further improve the confidence by which carbonylated peptides are identified via mass spectrometry, a novel 18O-labeling method that selectively introduces an 18O molecule into the carbonyl oxygen of carbonylated peptides was developed. The resulting 18O isotope signature enables carbonylated peptide precursor ions and carbonylated MS/MS fragment ions to be tracked in the full-scan MS and MS/MS spectra, respectively, thus providing an independent validation of the MS/MS spectra matched to carbonylated peptides by proteomic database searching. The value of 18O-labeling for both improving the accuracy and measuring the efficiency of database-based identification of carbonylated peptides is demonstrated in an HNE-treated lysate from rat skeletal muscle homogenate. In conclusion, the development of the SPH reagent and the 18O-labeling method are useful tools for identifying carbonylated peptides in complex biological mixtures and represent important steps forward in the field of redox proteomics.
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    Informing the Oral Squamous Cell Carcinoma Biomarker Search by Exudate Proteomics
    (2013-04) Kooren, Joel Allan
    Oral cancer is the sixth most common cancer worldwide ahead of Hodgkin's lymphoma, leukemia, brain, stomach, or ovarian cancers, with about 41,000 Americans being diagnosed annually. More than 90% of oral cancers are oral Squamous cell carcinomas (OSCC). While the overall 5-year survival rate is about 60%, the survival rate when diagnosed early is higher than 80%. Currently the standard for diagnosis of OSCC is early visual detection of a suspicious oral lesion followed by scalpel biopsy with histology. However, the invasiveness, expense, and required expertise involved prevents consistent application on at risk individuals. Chapter 1 discusses the methods that are being investigated for sampling and discovering biomarkers of OSCC that address some of these limitations. Protein biomarkers contained in samples collected non-invasively and directly from at-risk oral premalignant lesions (OPML) would address current needs in a uniquely targeted fashion. Chapter 2 of this thesis describes work evaluating the potential of a novel method using commercial PerioPaper absorbent strips for the collection of oral lesion exudate fluid coupled with mass spectrometry based proteomics for OSCC biomarker discovery. This research focuses on demonstrating the feasibility of using oral lesion exudates in proteomic research, exploring the proteome of exudate samples, discriminating between exudates collected from clinically different sources, with supplemental table 1 showing which proteins distinguish healthy and OPML sources. Furthermore, to ensure that the best possible marker candidates are selected given clinical sample availability, multiple methods were explored enable and improve quantitative proteomic analysis of exudates in chapter 3 (Identified proteins in supplemental files 2 and 3). Our label-free quantitative proteomics strategy analyzed paired control and OPML exudates (figure 8), identifying differentially abundant proteins between sample types. Next, we selected several [exudate] differentially abundant proteins for testing in while saliva, comparing their relative abundance levels in healthy, OPML and oral Squamous cell carcinoma (OSCC) subjects. Two proteins, CK10 and A1AT, showed differences in saliva. Our results provide a demonstration of the value of tissue exudate analysis for guiding salivary biomarker discovery in oral cancer, as well as providing promising biomarker candidates for future evaluation.
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    Infrared Spectroscopy of Gas Phase Ions: Using Infrared Multi-photon Spectroscopy to Investigate Ionization Sites and Hydrogen Bonding in Gaseous Ions
    (2013-07) Schmidt, Jacob C.
    In this work a novel instrument was developed to obtain infrared multiphoton dissociation (IRMPD) spectra of gas phase ions. This was done by coupling a tunable infrared laser with a Fourier transform mass spectrometer (FTMS). Gas phase ions were trapped in the FTMS cell and irradiated with infrared light for a period of time, typically 1-60 seconds, and the fragmentation of the parent ions was observed. Fragmentation was plotted vs. infrared wavelength to generate infrared action spectra. Experimental spectra were compared to computational spectra to gain insight into the gas phase structure of the irradiated ions. Some species were then derivatized to experimentally assign infrared absorption bands. The first objective was the determination of the ionization site of the (M+H)+ ion of p-aminobenzoic acid (1) formed during positive mode electrospray ionization (ESI) from either methanol/water or acetonitrile solutions. The two most likely protonation sites are at the amino or carboxyl functional groups. Protonated p-aminobenzoic acid ions were irradiated for 20 seconds from 2800-4000 cm-1. Computational predictions for both amino-protonated and carboxy-protonated ions were generated. When methanol/water was used as the ESI solvent there was an excellent match between experimental and the carboxy-protonated computational spectra, indicating that the carboxy-protonated structure was generated. When acetonitrile was used as the ESI solvent the experimental spectrum is identical to the carboxy-protonated structure, however, only 70% of the ions fragment. This indicates that a 70 : 30 ratio of carboxy : amino protonated structures were generated. A series of poly-hydroxy hydrocarbons (polyols) were investigated via IRMPD. Of particular interest is the extent of intramolecular hydrogen bonding in polyols containing as many as seven hydroxyl groups (i.e. 2), as well as the binding characteristics of a series of anions to triol 3. The infrared action spectrum of polyol 2 clustered with chloride anion shows the presence of several hydroxyl groups with varying hydrogen bond strengths indicating that a second, and even third, solvation shell forms around the chloride anion. Chlorine and sulfur anions prefer a geometry with the maximum of three hydrogen bonds while oxygen anions are presumably too small and thus only utilizes two of the three possible hydrogen bonds. Finally, the ionic structure of proline clustered to chloride anion was examined by IRMPD. Whether proline exists in its zwitterionic or neutral structure is not well understood. An exhaustive computational analysis was conducted on multiple neutral and zwitterionic conformers and the computed spectra were compared to the experimental infrared action spectrum. Furthermore, infrared action spectra of proline-d2 and proline-d7 were obtained to empirically assign infrared absorption features. It was determined that while the observed IRMPD spectrum suggests the neutral structure was generated as the sole ion, a more in depth study of the photokinetic data and computations indicate that both the neutral and zwitterionic structures are generated in approximately equal proportions.
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    Investigation of proteins that interact with NAADP-Gated two-pore channels.
    (2012-02) Moshier, Yaping Lin
    All living organisms respond to environmental stimuli by eliciting a sequence of signaling cascades, many of which converge in regulating [Ca2+]cyt via intracellular Ca2+ stores. Three agonist-mediated second messengers have been identified, including inositol 1,4,5-trisphosphate (IP3), cyclic ADP ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). NAADP is the most potent calcium mobilizer identified to date, and unlike IP3 and cADPR that target ER Ca2+ stores, NAADP-mediated Ca2+ response is restricted to acidic Ca2+ stores. Several candidate Ca2+ channels expressed in the endolysosomal system have been proposed to be gated by NAADP, with recently two-pore channels (TPCs) emerging as NAADP targets. My research project utilized a radioactive photoactivable NAADP analogue, 32P-5 azido-NAADP (32P-5N3-NAADP), to perform an unbiased assay in a variety of model systems, attempting to verify NAADP targets via a direct crosslinking approach. My results revealed that 5N3-NAADP labeled protein candidate(s) were significantly smaller than predicted sizes of TPC proteins (85-100kDa) in all systems examined (22/23kDa doublet in mammals, 41kDa in sea urchin). Further, the labeling pattern and intensity of the NAADP-targeted doublet remained unchanged in TPC-overexpressing cells. Surprisingly, photoaffinity labeling (PAL) of the high affinity NAADP targets was preserved in TPC-knockout pancreatic samples, further suggesting that NAADP binding and Ca2+ release are mediated by distinct protein identities. My data contradicts current models that TPCs are direct NAADP receptors, implying an alternative cellular binding partner for NAADP on endolysosomes that then serves to indirectly regulate TPC activity. This is an important revision of current dogma, and crucial for rational design of drugs that may modulate NAADP activity. Such therapeutics may be important in disorders (diabetes, lysosomal storage disorders, and neuronal excitotoxicity) where NAADP signaling is pathologically perturbed.
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    Mass Spectrometry Based Quantification of 1, 3-Butadiene Induced DNA Adducts: Potential Biomarkers of Cancer Risk
    (2014-11) Sangaraju, Dewakar
    Chemical carcinogenesis involves metabolic activation of carcinogens to electrophilic species which can react with important cellular biomolecules including DNA to form covalent adducts. Covalent carcinogen-DNA adducts which are not removed by DNA repair mechanisms can induce transforming mutations, ultimately leading to cancer. Hence, carcinogen-DNA adducts are deemed the ultimate biomarkers of carcinogen exposure, metabolic activation, and possibly of cancer risk. 1,3-Butadiene (BD) is a recognized human and animal carcinogen present in cigarette smoke, automobile exhaust, wood fires, and also in some occupational settings such as BD monomer and polymer plants. BD is metabolically activated by CYP2E1 to form three electrophilic epoxides: 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, EBD, and DEB can modify DNA bases to form covalent DNA adducts such as N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII), N7-(2, 3, 4-trihydroxybut-1-yl)-guanine (N7-THBG) and 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD). Although BD-DNA adducts had been successfully detected and quantified in tissues of laboratory animals exposed to relatively high concentrations of BD ( ≥ 6.25 ppm), they had not been previously quantified in humans, preventing their use as biomarkers of BD exposure, metabolic activation, and cancer risk. The main purpose of this research was to develop ultra-sensitive bioanalytical methodologies based on mass spectrometry to enable the detection and quantitation of BD-DNA adducts in animals treated with sup-ppm levels of BD and in exposed human populations. In Chapter 2 of the thesis, a novel nanoHPLC-nanoESI+-MS/MS method was developed for sensitive, accurate, and precise quantitation of BD-induced guanine-guanine cross-links (1,4-bis-(guan-7-yl)-2,3,-butanediol, bis-N7G-BD) in tissues of laboratory mice treated with low - sub-ppm concentrations of BD (0.5-1.5 ppm) which approximate human occupational exposure to BD (1 ppm). Bis-N7G-BD concentrations increased in a concentration-dependent manner in mouse liver DNA as a function of BD exposure. In Chapter 3 of this Thesis, we investigated DNA repair mechanisms responsible for bis-N7G-BD repair using isogenic Chinese hamster lung fibroblasts proficient or deficient in nucleotide excision repair (NER) and Fanconi Anemia (FA) repair pathways. We found that while both pathways contributed to bis-N7G-BD removal, FA pathway was most effective at alleviating the toxicity and replication blockage imposed by bis-N7G-BD cross-links. To enable BD-DNA adduct detection in humans, we developed an isotope dilution capillary HPLC-ESI+-HRMS/MS methodology for the most abundant BD-DNA adducts identified in vivo: N7-(2,3,4-trihydroxybut-1-yl)-guanine (N7-THBG) (Chapter 4). This method was successfully applied to quantify N7-THBG adducts in blood leukocyte DNA of smokers, nonsmokers, and occupationally exposed workers. In addition, we have developed an isotope dilution nanoLC/ESI+-HRMS3 methodology for the quantitation of BD-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human blood leukocyte DNA and human urine (Chapters 5 and 6). This method was applied to quantify EB-GII adducts in blood and urine of BD-exposed populations such as smokers, nonsmokers, and occupationally exposed workers. Overall, during the course of the studies described in this Thesis, we have developed a range of novel mass spectrometry-based quantitative methods which have excellent sensitivity, accuracy, and precision, and can be used for future human BD exposure biomonitoring studies. Furthermore, these methodologies are now being employed in epidemiological studies to identify any ethnic/racial differences in BD bioactivation and to help understand the origins of ethnic/racial differences in lung cancer risk in smokers.
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    Mass spectrometry-based analysis of urinary metabolites of 1,3-Butadiene (BD) in humans and influence of BD-DNA adducts on DNA replication
    (2013-10) Kotapati, Srikanth
    Cigarette smoking is a known risk factor for the development of lung cancer: approximately 1 out of 5 heavy smokers will develop the disease. However, there are significant differences in risk of lung cancer among smokers from different ethnic/racial groups. African American and Native Hawaiian smokers are at a higher risk of lung cancer than European American, Japanese American or Latin American smokers. Cigarette smoke has more than 70 known carcinogens. Following metabolic activation to electrophilic species, these carcinogens can form covalent DNA adducts, which are capable of inducing heritable mutations ultimately resulting in lung cancer. It has been hypothesized that the observed ethnic/racial differences in lung cancer risk in smokers are due to different frequencies of specific polymorphisms in drug metabolizing genes, leading to a different degree of carcinogen bioactivation to DNA-reactive intermediates. 1,3-Butadiene (BD) is among the most abundant and potent carcinogens present in cigarette smoke. BD is metabolically activated primarily by CYP2E1 to form 3,4-epoxy-1-butene (EB), hydroxymethyl vinylketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, HMVK, EBD, and DEB have been shown to modify DNA bases to form promutagenic adducts. Alternatively, EB, EBD, and DEB can undergo detoxification via epoxide hydrolysis (the main pathway in humans) or glutathione conjugation and further metabolic conversion into urinary mercapturic acids, 1-hydroxy 2-(N-acetylcysteinyl)-3-butene (MHBMA), 1,2-dihydroxy-4-(N-acetyl cysteinyl)-butane (DHBMA), 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA), respectively. The research presented in this thesis focuses on revealing any ethnic/racial differences in metabolism of BD in smokers and examining the ability of BD-DNA adducts to cause mutations. In the first part of the thesis, we have identified two novel metabolites of BD which have not been previously detected in vivo: 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA). To enable their detection in smokers, sensitive and specific HPLC-ESI--MS/MS methods were developed for both metabolites in human urine. We observed significant amounts of THBMA in samples from smokers, non-smokers and occupationally exposed workers. In contrast, bis-BDMA amounts in urine of smokers and occupationally exposed workers were below the method's limit of detection, although it was found in urine of F344 rats exposed to 62.5 or 200 ppm BD. Additionally we found significant interspecies differences in BD metabolism between laboratory rats and humans. DHBMA accounted for only 47% of BD urinary mercapturic acids in rats while the corresponding percentage in humans is 93%. We further developed a high throughput HPLC-ESI--MS/MS method for the quantification of MHBMA and DHBMA in humans and applied this method to quantify urinary BD-mercapturic acids metabolites in workers from a BD and styrene butadiene rubber (SBR) manufacturing facility and smokers belonging to different ethnic groups in two separate multi-ethnic cohort studies. Workers occupationally exposed to BD excreted significantly more BD-mercapturic acids than administrative workers at the same plant. In a small multi-ethnic study of smokers belonging to European American, Native Hawaiian and Japanese American (N = 200 per group), mean urinary MHBMA and MHBMA/DHBMA+MHBMA metabolic ratio were highest in European American and lowest in Japanese American smokers. Similar results were obtained in the larger study (N = 450 per group) composed of European American and African American smokers. Urine of European American smokers contained higher concentrations of MHBMA than that of African Americans. Genome-wide association study (GWAS) analysis conducted for the larger multi-ethnic group has revealed significant associations between single-nucleotide polymorphisms (SNPs) in chromosome 22 (22564172bp - 22735492 bp, nearby genes GSTT1, GSTT2, DDT and MIF) and urinary BD-mercapturic acid levels in smokers, providing the first evidence for genetic and ethnic/racial differences in metabolism of BD.The second part of my thesis work has focused on evaluating the mutagenic ability of three recently discovered BD-dA lesions: N6-(2-hydroxy-3-buten-1-yl)-adenine (N6-HB-dA), N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA), and 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-&gamma-HMHP-dA). In vitro translesion synthesis experiments were performed on synthetic oligonucleotides containing each of the three lesions at a site-specific position by gel electrophoresis and HPLC-MS/MS. We found that human translesion synthesis (TLS) polymerases hPols &eta, &kappa, &iota and human polymerase &beta were able to bypass (S)-N6-HB-dA in an error-free manner because of the conserved Watson-Crick base pairing with dT. However, replication past both (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA lesions by TLS polymerases hPols &eta and &kappa was highly error-prone, resulting in A&rarrT, A&rarrC mutations and frameshift deletions. This is the first study that identifies (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA as BD-DNA adducts potentially responsible for the induction of A*rarrT mutations by BD.
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    Mass Spectrometry-Based Characterization, Quantitation, And Repair Investigations Of Complex DNA Lesions
    (2018-03) Groehler IV, Arnold
    DNA is constantly under the threat of damage by various endogenous and exogenous agents, leading to the structural modification of nucleobases (DNA adducts). These DNA adducts can range from smaller nucleoside monoadducts and exocyclic adducts, to the helix distorting and super-bulky DNA-DNA cross-links and DNA-protein cross-links. If not repaired, DNA adducts can inhibit crucial biological processes such as DNA replication, leading to adverse consequences such as mutagenesis and carcinogenesis. Therefore, understanding the atomic connectivity, extent of formation, and repair of DNA adducts is crucial to fully elucidating the biological consequences of the adduct. DNA-protein cross-links (DPCs) are ubiquitous, super-bulky DNA lesions that form when proteins become irreversibly trapped on chromosomal DNA. The structural complexity of cross-linking and the diversity of proteins susceptible to DPC formation represents significant challenges to studying the biological consequence of these adducts. In the first part of the thesis, we identified the protein constituents, structural characterized and quantified, and investigated the repair mechanism of bis-electrophile (Chapter 2) and reactive oxygen species (ROS, Chapters 3 and 4)-induced DPCs. In Chapter 2, we investigated DPC formation after exposure to N,N-bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-ethylamine (nornitrogen mustard, NOR), the two biologically active metabolites of the antitumor agent cyclophosphamide. A mass spectrometry-based proteomics approach was employed to characterize the protein constituents of PM- and NOR-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. HPLC-ESI+-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N-7 position of guanines in DNA. A sensitive and accurate Cys-NOR-N7G isotope dilution tandem mass spectrometry assay was developed to quantify PM-induced DPC formation and repair in mammalian cells proficient or deficient in a DNA repair pathway. In Chapters 3, we employed the model of left anterior descending artery ligation/reperfusion surgery in rat to show that ischemia/reperfusion injury is associated with the formation of hydroxyl radical-induced DNA-protein cross-links (DPCs) in cardiomyocytes. Mass spectrometry based experiments revealed that these conjugates were formed by a free radical mechanism and involved thymidine residues of DNA and tyrosine side chains of proteins (dT-Tyr). Quantitative proteomics experiments utilizing Tandem mass tags (TMT) revealed that radical-induced DPC formation increase after LAD-ligation/reperfusion compared to the control sham surgery. Using the developed dT-Tyr nanoLC-ESI+-MS/MS assay, we investigated the role of the metalloprotease Spartan (SPRTN) in the repair of radical-induced DPCs (Chapter 4). Analysis of the brain, liver, heart, and kidneys of wild type (SPRTN+/+) and hypomorphic (SPRTN f/-) mice revealed a 1.5 – 2-fold increase in dT-Tyr in the hypomorphic mice, providing direct evidence that Spartan plays a role in the repair of radical-induced DPCs. Finally, we investigated the formation of formamidopyrimidine (FAPy) adducts after exposure to 3,4-epoxybutene, an epoxide metabolite of the known carcinogen 1,3-butadiene (Chapter 5). We successfully synthesized and structurally characterized a novel BD-induced DNA adduct EB-FAPy-dG, and developed a sensitive isotope dilution tandem mass spectrometry assay for its detection in vitro and in cells. To our knowledge, this is the first report of a BD-induced FAPy adduct, and future studies will examine whether BD-induced FAPy adducts In summary, during the course of this Thesis, we utilized mass spectrometry-based proteomics techniques to identify the proteins susceptible to PM- and ROS-induced DPC formation. After structurally characterizing the atomic connectivity of these adduces, we developed sensitive and accurate isotope dilution tandem mass spectrometry assays to perform absolute quantitation of PM- and ROS-induced DPC formation in cells and tissues. These assays were further utilized to begin investigating the repair mechanism of DPCs in cells and tissues, including providing direct evidence that the metalloprotease Spartan is involved in the repair of radical-induced DPCs. Finally, we detected EB-FAPy-dG formation in vitro and in vivo, the first evidence of 1,3-butadiene induced formamidopyrimidine formation.