The LC/MS/MS Analysis of Pyridinoline and Desmosines in Hypertensive Mouse Aorta, Elucidation of Arginine and Proline Fragmentation for the Analysis of Arginine Metabolism in Mosquitoes by LC/MS/MS, and Mudpit Identification of B. Pseudomallei Proteins in Urine

Abstract

This dissertation presents an investigation into the application of mass spectrometry to the detection and quantitation of proteins and amino acids in complex biological samples. This is accomplished by two dimensional chromatography (strong cation exchange / reverse phase) coupled to tandem mass spectrometry followed by peptide spectrum matching for the detection of Burkholderia pseudomallei proteins in infected patient urine samples and reverse phase liquid chromatography coupled to a triple quadrupole and quadrupole time of flight mass spectrometers for the quantitation of cross-linked or free amino acids in mouse aorta or mosquito excreta, respectively. B. pseudomallei is a pathogenic gram negative bacillus that is endemic to the populations of Southeast Asia and is the causative agent of the disease melioidosis. LC/LC/MS/MS was used to identify candidate B. pseudomallei proteins for the development of a lateral flow immunoassay feasible for use in the impoverished communities that melioidosis affects. Three proteins (GroEL, FliC, and BipC) were identified, and have been detected in western blots and ELISAs of patient urine. Angiotensin II is known to increase both hypertension and vascular and cellular extracellular matrix remodeling, but little is known about the underlying mechanism of angiotensin II action on ECM remodeling. It has been hypothesized that the cross-linking of collagen by the enzyme lysyl oxidase increases the stiffness of the vasculature as increased levels of lysyl oxidase expression and activity have been observed in angiotensin II models of hypertension. LC/MS/MS was used to show that the cross-linking of ECM proteins increased over time in mice treated with angiotensin II over 4 weeks using a novel method to account for tissue heterogeneity in mouse aorta samples. Mosquitoes are missing a key enzyme in the urea cycle which makes arginine both an essential amino acid for mosquitoes but also makes mosquitoes unable catabolize arginine into non-toxic metabolites. MS/MS was used to show that mosquitoes excrete high levels of arginine after feeding. In addition, a previously undescribed fragmentation of arginine was elucidated using ¹⁸O labeling for future metabolism studies that would require the determination of individual arginine carbons based on fragmentation spectra.