Doctor of Philosophy (PhD)
This dissertation describes the integration of micro-scale separations to matrix assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI TOF MS) for protein analysis. MALDI MS provides unsurpassed accurate mass measurements of intact bio-molecules, for example peptides and proteins, which in turn generate high molecular specificity enabling the identity, function and structure of these molecules to be characterized. However, in order to realize the full potential of MS in proteomic studies, integrated sample processing on automated and high throughput platforms is required to address the complexity, diversity and the dynamic range of proteomic analysis. The work described here contributes towards the development of automated and high throughput micro-total analysis systems (µ-TAS) for proteomics. An overview of mass spectrometry instrumentation and techniques used in protein analysis is presented to highlight the significance of the work described. Microfluidics devices can serve as automated and high throughput platforms for integrating proteomics sample processing steps such as whole cell lyses, enrichment, solubilization, denaturation, protein separations, proteolytic digestion and chromatographic separations of peptides prior to MALDI TOF MS analysis. Therefore, coupling microfluidics devices to biological mass spectrometry is the first logical step towards developing fully integrated and automated systems for protein analysis. On-line and off-line approaches for analysis from microfluidic devices are discussed. The development of a specially tailored rotating ball inlet for automated on-line MALDI MS sample introduction from an electrophoresis-based separation platform is described. Electrophoresis-based micro-scale separations of peptides on fused silica capillary and polymer-based microfluidic devices were coupled to on-line MALDI TOF MS using a rotating ball inlet. The rotating ball inlet allowed for individual technique optimization and automation thereby eliminating the need for fractionation and routine MALDI sample preparation. High throughput solid phase micro-reactors for efficient enzymatic cleavages and improved protein identification with MALDI MS in a microfluidic device were also developed for incorporation in an integrated protein analysis microfluidic system. Future work that outlines the framework and focus geared towards integrating the modules discussed in this dissertation into a functional micro-total analysis system for protein sample processing is discussed.
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Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.
Musyimi, Harrison K., "Integrating Micro-Scale Separations to Matrix Assisted Laser Desorption and Ioniation Time of Flight Mass Spectrometry (MALDI-TOF-MS) for Protein Analysis" (2006). LSU Doctoral Dissertations. 810.
Steven A. Soper