Browsing by Author "David C. Muddiman, Committee Chair"

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Development and Characterization of an Atmospheric Pressure Ionization Source Matrix-Assisted Laser Desorption Electrospray Ionization Coupled to Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Analysis of Biological Macromolecules
(2009-07-16) Sampson, Jason Scott; Jorge A. Piedrahita, Committee Member; Edmond F. Bowden, Committee Member; Kenneth W. Hanck, Committee Member; David C. Muddiman, Committee Chair
The field of mass spectrometry has grown tremendously over the past few decades due in large part to the continued application to new and interesting areas of exploration. The advent of soft ionization sources such as electrospray ionization and matrix-assisted laser desorption/ionization have dramatically increased the capabilities of mass spectrometry and increased the amount of information derived from biological samples. As a result, there has been tremendous growth in the development of new ionization source technology in recent years. One of the main driving forces behind the development of new ionization technology is for the reduction of sample preparation required prior to analysis, yielding high throughput sample analysis. Demonstrated herein is the development and characterization of an atmospheric pressure ionization source called matrix-assisted laser desorption electrospray ionization (MALDESI). MALDESI is a hybrid combination of MALDI and ESI which utilizes laser desorption with electrospray postionization for the generation of multiply-charged ions. Multiply-charged ions are of particular importance when using Fourier transform mass spectrometry, due to the increase in resolving power and mass accuracy with increasing charge on the molecule. Top-down characterization of intact polypeptides is demonstrated as well as high mass accuracy utilizing internal calibration. A newly designed highly robust and versatile atmospheric pressure ionization platform is developed and described in detail. Solid- and liquid state analysis of three out of the four classes of biological molecules is demonstrated utilizing the newly developed versatile ionization platform. MALDESI utilizing ultraviolet and infrared laser desorption is demonstrated at various wavelengths (UV, 337 nm and 349 nm and IR, 2.94 Ã‚Âµm and 10.6 Ã‚Âµm) with and without ESI postionization for the generation of multiply-charged ions. The characteristics of liquid-state samples are described as a macroscopic electrospray droplet and characterized. MALDESI direct analysis is demonstrated and applications for high throughput analysis of complex samples are described.
The Development and Utilization of Aerodynamic Devices in Biological Mass Spectrometry
(2009-09-03) Dixon, Robert Brent; Edmond Bowden, Committee Member; Jack R. Edwards, Committee Member; Kenneth Hanck, Committee Member; David C. Muddiman, Committee Chair
The role of mass spectrometry in the chemical and biological sciences is ever evolving as there is an increasing interest to understand the biomolecular structure and interaction of proteins and their potential impact on human health. To this end, emerging techniques that complement or enhance mass spectrometric capabilities are of significant relevance. While the impact of mass spectrometry in every field (e.g. proteomics, drug development, environmental, crop science, or petrochemical) has varied, increased accessibility of instrumentation and expertise to address relevant issues across disciplines will result in even greater acceptance of this powerful technology. Some compounds may not ionize by a predominant method, electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI), but recently a number of ionization sources have been developed that further encompass more classes of molecules for mass spectrometric analysis. Limited sample availability is a practical issue which demands lower limits-of-detection. In efforts to address this, devices such as the ion funnel and the air amplifier are being developed to more efficiently transport ions within or into the mass spectrometer (respectively). With continued ionization source and instrumentation improvements, there will undoubtedly be additional physicians, scientists, and engineers who realize the great potential and benefit of using mass spectrometry to address questions related to their particular field of study. This dissertation describes the utilization of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which affords high resolving power and mass measurement accuracy, for the majority of ionization source developments. These ionization source efforts have been directed at improving current and emerging ionization techniques. A method for transporting neutral species for subsequent ionization termed remote analyte sampling, transport and ionization relay (RASTIR) has been developed and is described herein. Improvements for electrospray ionization have been realized using the air amplifier as it focuses and desolvates electrospray droplets; these mechanisms for improved ion abundance are investigated. An acoustic method for direct analysis of proteins and peptides from an oscillating surface is the premise of the radio frequency acoustic desorption ionization source. Collectively, this work aims to improve existing ionization techniques while investigating new avenues for biological mass spectrometry.
Exploring Fundamental Aspects of Proteomic Measurements: Increasing Mass Measurement Accuracy, Streamlining Absolute Quantification, and Increasing Electrospray Response
(2009-05-08) Williams, Dennis Keith Jr.; Kenneth W. Hanck, Committee Member; Edmond F. Bowden, Committee Member; David C. Muddiman, Committee Chair; Lin He, Committee Member
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers unparalleled performance in terms of resolving power and mass measurement accuracy. In order to realize the highest achievable mass measurement accuracy for a given FT-ICR MS system, frequency shifts due to space-charge effects must be accounted for via external or internal calibration methods. Herein, a dual electrospray ionization source was coupled to a hybrid quadrupole Fourier transform ion cyclotron resonance mass spectrometer and utilized to incorporate internal calibrant to yield high mass measurement accuracy results. Frequency shifts from space-charge effects were also counteracted by accounting for total ion population and relative ion population on a MALDI-FT-ICR MS and a hybrid LTQ-FT-ICR MS equipped with automatic gain control. This was achieved through the use of multiple linear regression and artificial neural networks. These experiments resulted in mean mass measurement accuracies in the parts-per-billion range. C-reactive protein (CRP) is an important clinical marker for inflammation, atherosclerosis, and has also been observed to be upregulated in patients with epithelial ovarian cancer (EOC). This dissertation contains results from a protein cleavage isotope dilution mass spectrometry method developed for the absolute quantification of CRP from human plasma. A total of 110 human plasma samples were analyzed including 54 samples from patients with EOC. The results were compared to a CLIA certified ELISA test which showed high correlation but different absolute values, which suggested different reference ranges for different analytical techniques. In addition, a correlation was observed between the stage of diagnosis of cancer and CRP concentrations. Chemical tags have long been utilized with mass spectrometry for a multitude of purposes, extending the effectiveness of the measurements dramatically. Electrospray ionization has been shown to preferentially ionize more hydrophobic species. Four new iodoacetamide derivatives, which react with the amino acid cysteine, were reacted with three peptides to determine their ability to increase electrospray response. This resulted in increases up to 2000-fold compared to alkylation of peptides with iodoacetamide. The combination of these results will aid future studies to identify and quantify proteins and peptides which contain cysteine and can be expanded to all peptides using amine-specific chemistry.