Date of Award
Doctor of Philosophy
Michael J. Sepaniak
S. Douglass Gilman, Mark Dadmun, Youngmi Lee, Robert Hettich
Though the study of the human proteome (proteomics) is far more complicated than that of the genome, it may prove to be the next step toward gaining insight into the operation of biological systems. Understanding the role proteins and peptides play in disease, whether that be by their structure, expression or function, could lead to the identification of disease biomarkers for diagnosis or engender possibilities for treatment. To this end, the work discussed herein delves into the capillary electrophoretic (CE) analysis of amyloid beta conformations involved in abnormal aggregation and the creation and optimization of a novel bioassay method. By rapidly analyzing the conformations of Aβ present in heterogeneous mixtures, valuable insight may be provided into the mechanism of abnormal protein aggregation. Further, the novel bioassay described herein may supply a simplified method for analyzing and quantifying many different biologically relevant interactions (i.e. DNA hybridization and immunoassays).
Capillary electrophoresis is used to study the aggregation pathway of the amyloid beta peptide (Aβ). Since little is known about the mechanism of Aβ fibril formation, this research sought to rapidly characterize different conformations of Aβ present in a heterogeneous mixture of species using changes in effective mobility and peak shape during CE separation. Electron microscopy and mass spectrometry were used to confirm the presence of Aβ in the mixtures and collected peak fractions. Additionally, the interaction between wild type Aβ and a mutated Aβ species resulting in increased protofibril formation was analyzed using CE.
A novel bioassay method was also developed and termed magnetically assisted transport evanescent field fluoro-assays (MATEFFs). MATEFFs employ magnetic beads as both a solid phase for analyte pre-concentration and delivery method to a localized evanescent field. Using this technique, the measurement of signal can be performed without call for wash steps and while avoiding matrix interferents present in bulk sample solution. While not limited by analyte diffusion, this method still allows for targeted delivery using simple magnetic control of analyte-labeled beads. This could impact the bioassay field, as much focus is currently directed toward miniaturization of bioassay formats.
Wellman, Amber D., "Innovative Methods of Biomedical Molecular Analysis: Capillary Electrophoretic Study of Amyloid Beta Conformations and Magnetically Assisted Transport Evanescent Field Fluoro-assays. " PhD diss., University of Tennessee, 2007.