Altering Nitric Oxide Bioavailability and Lipid Profiles in Endothelial Cells By Polycyclic Aromatic Hydrocarbons in Particulate Matter

Epidemiologic studies have demonstrated a significant association between exposure to particulate matter (PM) and atherosclerosis. Polycyclic aromatic hydrocarbons (PAHs) present in particulate matter, are well known to induce oxidative stress and lipid peroxidation via generation of reactive oxygen species (ROS). Lipid peroxidation involves regulating endothelial nitric oxide synthase via inhibition of its activity, and as a result, mediates dilation of coronary arterioles is involved with the pathogenesis of atherosclerosis. However, data on assessment of oxidized lipid formation is limited due to low resolution of mass spectrometer methods. Taking the advantage of a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS) with 12 Tesla at Old Dominion University, this study assessed how lipid peroxidation induced by PAHs altered the lipid profile and nitric oxide level in human endothelial cells. Human coronary artery endothelial cells (HCAEC) were exposed to PAHs with various doses and treatment duration times. After exposure, the level of ROS was measured using the fluorometric method with a flow cytometer. Lipid peroxidation was assessed based on the formation of malondialdehyde. Nitric oxide synthase was calculated using a cell-permeable diacetate that reacts with NO to form a fluorescent triazolotluorescein. Then, lipids of HCAEC were extracted by chloroform and methanol and analyzed using 12T FT-ICR-MS to separate peaks. After PAH exposure, cell morphology noticeably changed, granularity increased, and viability decreased after the short term treatment. The ROS level, expressed as fluorescent intensity readings from a flow cytometer, significantly increased along with the malondialdehyde levels. The analysis of spectrum data from FT-ICR-MS showed changes of the cellular lipid profile in the exposed groups as compared to the control. We found no significant impacts of oxidative stress on nitric oxide bioavailability in endothelial cells. However, direct exposure of HCAEC to PAHs increased lipid oxidization. These data imply that PAHs may mediate nitric oxide synthase activity through lipid peroxidation by a mechanism yet to be elucidated. These studies demonstrate one potential means by which oxidative stress can induce endothelial cell damage and lipid peroxidation during atherosclerosis for a short term exposure. In conclusion, the oxidation pathway induced by PAHs contributed to endothelial cell damage. This study identified lipids and developed novel data analytic approaches that are applicable in revealing oxidative lipid formation induced by PAHs. In addition, the technology enables us to identify specifically oxidized phospholipids that could serve as biomarkers in assessing PAH-induced endothelial dysfunction and the underlying mechanism

Matrix-assisted laser desorption/ionization mass spectrometry for the analysis of collected bioaerosols

The goal of this dissertation was to demonstrate collection, detection and identification of microorganisms from bioaerosols using offline matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of proteins. MALDI-MS intact bacteria techniques were adapted for use with an orthogonal MALDI quadrupole-time-of-flight mass spectrometer. Results indicate the instrument is capable of analyzing intact whole-cells. The first phase was to evaluate three bioaerosol samplers, an Andersen N6 single-stage impactor (AI), a cyclone impactor, and a vacuum filter system. The samplers collected test bioaerosols using a constructed bioaerosol exposure chamber (BEC). The BEC allowed all three samplers to operate in parallel. Each sampler demonstrated the ability to successfully collect and detect the test bioaerosol by offline MALDI-MS. Using the TOF-MS spectra from impacted bacteria, the Expert Protein Analysis System\u27s (ExPASy) sequence retrieval system (SRS) was used to search the SWISS-PROT database. A total of 19 unique proteins were identified for E. coli,8 for B. Thuringiensis, and 6 for B. subtilis. Subsequently, cytochrome c and E. coli samples were proteolyzed in situ using trypsin and CNBr. The digestions were done using removable mini-wells. The mini-wells were placed on top of collected spots on the MALDI target and served as a mini chemical reactor for digestion. Using the TOF-MS spectra of the digested samples, peptide mass mapping was done using the MASCOT search engine. A progressive reductive iterative search mapping (PRISM) technique was used in order to assist in optimizing protein matches from E. coli. In this approach, four of seven iterations produced protein matches. To determine the suitability of MS/MS techniques for use with in situ digests, selected fragments from the cytochrome c and E. coli digests was done. MS/MS was successful for cytochrome c, but was unable to produce spectra for E. coli

Novel concepts for lipid identification from shotgun mass spectra using a customized query language

Lipids are the main component of semipermeable cell membranes and linked to several important physiological processes. Shotgun lipidomics relies on the direct infusion of total lipid extracts from cells, tissues or organisms into the mass spectrometer and is a powerful tool to elucidate their molecular composition. Despite the technical advances in modern mass spectrometry the currently available software underperforms in several aspects of the lipidomics pipeline. This thesis addresses these issues by presenting a new concept for lipid identification using a customized query language for mass spectra in combination with efficient spectra alignment algorithms which are implemented in the open source kit “LipidXplorer”