Type of Document Dissertation Author Washington, Samuel Jean Author's Email Address firstname.lastname@example.org URN etd-04122007-183135 Title CHEMICAL DIFFERENCES OF ATHEROSCLEROTIC PLAQUES IN NATIVE AND BYPASS HUMAN CORONARY ARTERIES AND DISEASED AND NON-DISEASED HUMAN AORTAS Degree Doctor of Philosophy (Ph.D.) Department Chemistry Advisory Committee
Advisor Name Title Isiah Warner Committee Chair Patrick A. Limbach Committee Co-Chair George Stanley Committee Member Kermit Murray Committee Member Sue G. Bartlett Committee Member Thomas P. Gillis Dean's Representative Keywords
- mass spectrometry
- gel electrophoresis
Date of Defense 2007-03-27 Availability unrestricted AbstractABSTRACT
Cardiovascular disease, primarily atherosclerosis involves a number of distinct processes that are associated with plaque development. The use of differential gel electrophoresis to determine differences between proteins produced in native and bypass coronary arteries from the same heart has been studied. The analytical techniques presented in this study to characterize plaque samples include two dimensional gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, mass spectrometry, differential gel electrophoresis, and high performance liquid chromatography.
An overview of the stages involved in atherosclerosis and the theories implicated in the manifestation of atherosclerosis was presented. A design study using the Box Behnken method was used to optimize the components in lysis buffer to solubilize the membrane proteins present in the intima in atherosclerotic plaques. The number of proteins located as spots in the gel were optimized in this study.
The isolation and characterization of proteins in intima and media extracts of diseased aortas were separated using two dimensional gel electrophoresis followed by excision of gel spots and tryptic digestion to peptides. Mass analysis (MS and MS/MS) of the digested peptides with a linear quadrupole trap Fourier transform ion cyclotron resonance mass spectrometer was used to determine the amino acid sequences.
Protein extracts of normal and diseased aortas and native and bypass arteries were analyzed using differential gel electrophoresis system. Cydye 3 (1-(5-carboxypentyl)-1`-propylindocarbocyanine halide N-hydroxy-succinimidyl ester) and Cydye 5 (1-(5-carboxypentyl)-1´-methylindodicarbocyanine halide N-hydroxy-succinimidyl ester) are fluorescent dyes used in our study to identify differentially expressed proteins of interest in the same gel using matrix assisted laser desorption ionization time-of-flight mass spectrometry. Several proteins in normal and diseased tissues were identified
A study of the separation and quantification of cholesterol and cholesteryl esters was performed using reverse phase high performance liquid chromatograph interfaced with atmospheric pressure chemical ionization probe that was introduced into a quadrupole mass analyzer. Thirty-five samples were analyzed by principal component analysis that produced two distinct age groups based on age. In addition, a disease severity index was generated from the data. This study concluded that age and low disease severity index were indicators of the atherogenic states of the extracts analyzed.
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