Preeclampsia (PE), a life threatening pregnancy-related disorder, is characterized mainly by new onset of hypertension and proteinuria after 20 weeks of gestation. Currently, PE cannot be predicted prior to onset of symptoms and there is no cure for the disease. There is a clear value in having biomarkers able, early in a pregnancy, to identify women at risk for PE so that proper treatment therapies could be developed. Although a number of serum candidate markers have been proposed to be altered in PE patients, their use is limited due to poor sensitivity and specificity. Therefore, there is ongoing need for better set of novel biomarkers predicting PE. Consequently, for my first project, we used a serum proteomic approach involving reversed phase capillary-liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (cLC-ESI-QTOF). Our approach focuses on the less abundant (nM or lower), lower molecular weight peptides and lipids predicting PE. We got previously collected sera from pregnant women at 12–14 weeks gestation. There were 24 controls, having term uncomplicated pregnancies and 24 cases, which developed PE later in the same pregnancy. Many statistically significant serum PE biomarker candidates were found comparing cases and controls. In addition, multimarker combinations having high detection sensitivity and specificity (AUC >0.9) were developed using logistic regression analysis. For my second project, serum lipidomic analysis of sera from pregnant women was undertaken to determine if useful PE lipid biomarkers exist. A discovery study involving a shotgun lipidomic approach was performed using sera collected at 12-14 weeks of pregnancy from 27 controls with uncomplicated pregnancies and 29 cases that later developed PE. Lipids were extracted using organic solvent and analyzed by direct infusion into a time-of-flight mass spectrometer. Statistically significant lipid markers were found and reevaluated in a second confirmatory study having 43 controls and 37 PE cases. The initial study detected 45 potential PE markers. Of these, 23 markers continued to be statistically significant in the second confirmatory set. Several multi-marker panels with AUC >0.85 and high predictive values were developed from these markers. My third project also involved the above mentioned approach for detection of novel lipid biomarkers for Alzheimer's disease. Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of age-related dementia. Currently, there are no methods to detect Alzheimer's at an early stage when treatment therapies could be applied. Therefore, there is need for detection of panel of biomarkers for detecting patients at risk to AD at an early stage. In the initial discovery set, sera from 29 different stage AD cases and 32 controls were analyzed using direct infusion mass spectrometry (ESI-TOF). This study yielded 89 potential lipid biomarkers which were evaluated in another confirmation study. Of these, 35 markers continued to be statistically significant in the second confirmatory set. Using the confirmed markers, several multi-marker panels with AUC > 0.87 were developed for any stage AD cases vs controls. Multi-marker panels with AUCs > 0.90 were developed for each specific CDR vs controls, including the earliest stage of AD. These lipidomic biomarkers are likely to distinguish AD cases regardless of the stage from controls. In conclusion, we successfully detected, validated and identified low molecular weight novel biomarkers for PE and lipid biomarkers for AD.

Phospholipids (PLs) are a component of cell membranes, biological fluids and tissues. These compounds are problematic for the bioanalytical chemist, especially when PLs are not the analytes of interest. PL interference with bioanalysis highly impacts reverse-phase chromatographic methods coupled with mass spectrometric detection. Phospholipids are strongly retained on hydrophobic columns, and can cause significant ionization suppression in the mass spectrometer, as they out-compete analyte molecules for ionization. Strategies for improving analyte detection in the presence of PLs are reviewed, including in-analysis modifications and sample preparation strategies. Removal of interfering PLs prior to analysis seems to be most effective at moderating the matrix effects from these endogenous cellular components, and has the potential to simplify chromatography and improve column lifetime. Products targeted at PL removal for sample pre-treatment, as well as products that combine multiple modes of sample preparation (i.e. Hybrid SPE), show significant promise in mediating the effect on PL interference in bioanalysis.

N-Acylphosphatidylethanolamine (NAPE) and its hydrolysis product, N-acylethanolamine (NAE), are minor but ubiquitous lipids in multicellular eukaryotes. Various physiological processes are severely affected by altering the expression of fatty acid amide hydrolase (FAAH), an NAE-hydrolyzing enzyme. To determine the effect of altered FAAH activity on NAPE molecular species composition, NAE metabolism, and general membrane lipid metabolism, quantitative profiles of NAPEs, NAEs, galactolipids, and major and minor phospholipids for FAAH mutants of Arabidopsis were determined. The NAPE molecular species content was dramatically affected by reduced FAAH activity and elevated NAE content in faah knockouts, increasing by as much as 36-fold, far more than the NAE content, suggesting negative feedback regulation of phospholipase D-mediated NAPE hydrolysis by NAE. The N-acyl composition of NAPE remained similar to that of NAE, suggesting that the NAPE precursor pool largely determines NAE composition. Exogenous NAE 12:0 treatment elevated endogenous polyunsaturated NAE and NAPE levels in seedlings; NAE levels were increased more in faah knockouts than in wild-type or FAAH overexpressors. Treated seedlings with elevated NAE and NAPE levels showed impaired growth and reduced galactolipid synthesis by the “prokaryotic” (i.e., plastidic), but not the “eukaryotic” (i.e., extraplastidic), pathway. Overall, our data provide new insights into the regulation of NAPE–NAE metabolism and coordination of membrane lipid metabolism and seedling development.

Heart failure is a major public health issue, with its prevalence estimated to be 6.5 million adults in the USA. Of the hospitalized heart failure (HF) cases, 50% are characterized by preserved ejection function (HFpEF). In HFpEF, the heart pumps a normal proportion of blood that enters it. However, thickening of the ventricular walls inhibits the chamber filling to normal volume. The direct basis of HFpEF is a slowed elongation of the cardiac muscle during the diastolic phase of the cardiac cycle. Elucidation of mechanisms that mediate relaxation of cardiac muscle could help understand the pathogenic mechanisms in HFpEF. Myocardial contraction and relaxation are tightly controlled processes that involve thick and thin filament regulatory proteins. β-Adrenergic signaling pathway is a major regulator of myocardial contraction and relaxation via the activation of protein kinase A (PKA). Two key myofilament proteins, troponin I (TnI) and myosin binding protein-C (MyBPC), are phosphorylated by PKA following β-adrenergic stimulation. The purpose of this thesis is to develop a liquid chromatography-mass spectrometry (LC-MS) method for the quantification of phosphorylation in TnI and MyBPC and measure the changes in the degree of phosphorylation in transverse-aortic constriction (TAC) mouse hearts, a model representing HFpEF, and sham (control) mouse hearts. The initial approach of the project was to develop a method for quantification of phosphopeptides using synthesized stable isotope labeled (SIL) peptides, both with and without phosphate modification. To accomplish this goal, a multiple reactions monitoring (MRM)-LC-MS method for the quantification of the synthesized SIL peptides was first developed. This method, using low picomole amounts, is applicable to researchers in the field using SIL peptides for quantification. However, when the SIL peptides were actually applied, we determined that there was a selective absorption of some phosphate peptides in the LC column, limiting the use of the SIL peptides for quantification. This result is also of general interest to others trying to identify phosphopeptides, not realizing that some peptides will go unmeasured. Thus, we returned to expanding an earlier method developed in our research group to quantify the degree of phosphorylation. Key to this work was the development of a quantification method directly from heart myofibrillar protein preparations without requiring isolation of individual proteins by gel electrophoresis. Using the LC-MS method developed, we quantified phosphorylation sites of TnI and MyBPC in the TAC and control mouse hearts. The phosphorylation measurements showed no significant difference in phosphorylation between the TAC and control mice, except for one site, S302 in MyBPC that had a 13% decrease in phosphorylation with TAC. We conclude that in our TAC model, PKA dysfunction may not play a role in the initial development of HFpEF.

The characterization of biomolecules and biomolecular complexes represents an area of significant research activity because of the link between structure and function. Drug development relies on structural information in order to target certain domains. Many traditional biochemical techniques, however, are limited by their ability to characterize only certain stable forms of a molecule. As a result, multidimensional approaches, such as ion mobility mass spectrometry coupled to mass spectrometry (IMS-MS), are becoming very attractive tools as they provide fast separation, detection and identification of molecules, in addition to providing three-dimensional shape for structural elucidation. The present work expands the use and application of trapped ion mobility spectrometry-coupled to mass spectrometry (TIMS-MS) by analyzing a range of biomolecules (including proteoforms, intrinsically disordered peptides, DNA and molecular complexes). The aim is to i) evaluate the TIMS platform measuring sensitivity, selectivity, and separation of targeted compounds, ii) pioneer new applications of TIMS for a more efficient and higher throughput methodologies for identification and characterization of biomolecular ions, and iii) characterize the dynamics of selected biomolecules for insight into the folding pathways and the intra-or intermolecular interactions that define their conformational space.

Desorption electrospray ionization (DESI) is an ambient ionization technique used for mass spectrometric imaging of biological samples. When coupled with isotopic ratio measurements of deuterium-labeled tissues, DESI provides a means of measuring metabolic rates on a spatially resolved basis. In vivo metabolic rates are desired to better understand diseases such as Alzheimer's, Parkinson's, Huntington's, and various forms of cancer that negatively impact metabolic rates within different organs of the human body. Although DESI has been used to image lipids and metabolites of a variety of tissues and other imaging techniques, such as NIMS, have been used to study kinetic turnover rates, DESI has not yet been used to study in vivo metabolic rates using deuterium labeled tissue. This thesis describes how we optimized our DESI source for imaging of biological tissue, how we developed a MATLAB graphical user interface (GUI) to process and interpret the large mass spectral data files, how we conducted our initial mouse brain study for proof-of-concept, and how we plan to implement our DESI imaging in a study with mouse models of Alzheimer's disease. Our initial mouse brain study involved labeling mice with deuterium enriched water, preparing tissue slices for DESI analysis, imaging the tissue slices using DESI coupled with a Bruker mass spectrometer, analyzing the mass spectral data using our custom-designed image_inspector program, confirming identification of lipids using MS/MS, and creating incorporation curves to measure in vivo metabolic rates.

In the fight against disease and illness, nature has provided mankind some of our best therapeutics in the form of secondary metabolites. The plant, fungi and animal phyla inhabiting the Earth produce diverse and unique chemistry that can be used in our fight against disease. In the growing threat of drug resistance and pathogen evolution, the field of natural products chemistry strives to explore new biological and chemical diversity sources, and develop innovative methodology to identify and isolate new chemistry faster than ever. The dissertation herein presented is one such effort to find new, bioactive chemistry from the marine environments. New biodiversity sources, from the tropical Floridian mangrove forests to the cold waters of the Antarctic oceans, were evaluated for the new, unique chemistry they produce. A large-scale screening of epigenetically modulated mangrove fungi was undertaken, producing a large, biologically and chemically diverse extract library. New methodology was developed in order to evaluate these extracts, leading to rapid identification and isolation of known and new bioactive metabolites. From the Southern Oceans, a collection of sponges was studied, and a new, highly unique peptide was isolated and characterized. These efforts were undertaken in the continued effort to isolate new, unique lead compounds.

No description available.

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in μg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 μg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 μCi.mL-1, compared to a mean of 4.79 μCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 μg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 μg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 μg.mL-1, and a dexamethasone treated concentration of 22.84 μg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 μM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 μM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.

The physiologically important free radicals, nitrogen monoxide and superoxide, can combine to form the reactive intermediate peroxynitrite. Peroxynitrite can react with proteins and their constituent amino acids, such as tyrosine, resulting in protein peroxidation, oxidation and nitration. The nitration of proteins, assessed by the analysis of 3-nitrotyrosine, is a proposed index of pathophysiological activity of peroxynitrite. The aim of the work was to investigate the reaction products between peroxynitrite and protein, develop an assay for 3-nitrotyrosine and measure its levels in biological samples. To study the amino acid products arising from the reaction of peroxynitrite and protein, both liquid chromatography (LC) and gas chromatography (GC) combined with mass spectrometry (MS) were adopted. Approaches to 3-nitrotyrosine assay development were first, to take advantage of the intrinsic sensitivity of electron capture negative ionization GC-MS. Secondly, to avoid possible artefactual problems associated with the derivatisation step in GC-MS, an assay for 3-nitrotyrosine based on combined LC-MS-MS was developed. When a selection of peptides was exposed to peroxynitrite under physiological conditions in vitro, the hydrolysis products showed that 3-nitrotyrosine was the major product. Detectable minor products were 3,5-dinitrotyrosine and DOPA. The GC-MS assay was found to be fraught with difficulty due to artefactual formation of 3-nitrotyrosine. In order to quantify and correct for artefact formation, this complication was approached by incorporating a second isotopomer. This method, however, was confounded by large errors that reduced the overall sensitivity. Either negative or zero levels of endogenous 3-nitrotyrosine were found in tested samples after correction for artefact formation. The LC-MS-MS assay was then used to analyse 3-nitrotyrosine levels in a range of biological samples, including human plasma from healthy volunteers, synovial fluid samples from arthritis patients and tissue extracts from a mouse model of amyotropic lateral sclerosis. In contrast to published data, 3-nitrotyrosine levels were found to be below the limit of detection (1 pg/????L, 10 pg o/c) for all samples - a result somewhat consistent with the negative GC-MS data. It is suggested that the high 3-nitrotyrosine levels previously reported in the literature might reflect artefactual generation of 3-nitrotyrosine and that other approaches to assessing pathophysiological nitration should be sought in future.