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In this work, tandem mass spectrometry (MS/MS)-based quantitative protocols were developed to facilitate differential protein expression analysis and biomarker discovery via a two-step sample interrogation strategy: (a) global protein profiling and differential expression analysis by spectral counting; and, (b) biomarker candidate validation by targeted screening, i.e., multiple reaction monitoring (MRM). Preliminary experiments were performed to evaluate the performance of the spectral counting method. The method proved to be applicable for proteins with spectral counts≥2, and a close-to-linear relationship between protein concentration and spectral count data was achievable at protein concentration levels <0.1 μM. The detection limit was 40-800 fmol. A protein/peptide library containing ~10,000 peptide entries that facilitates the development of future MRM experiments, was developed. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum, the peptide retention time, and the top 10 most intense product ions that correspond to a given parent peptide. Only proteins identified by at least two spectral counts are listed. An MRM experiment was performed to demonstrate the successful applicability of this peptide library for the identification of putative biomarkers in proteomic samples. / Master of Science

Investigating the structure of proteins and their interactions with other biomolecules or drug molecules, coupled with the consideration of conformational change upon binding, is essential to better understand their functions. Mass spectrometry (MS) is emerging as a powerful tool to study protein and peptide structure and interactions due to the high dynamic range, low sample consumption and high sensitivity of this technique, providing insight into the stoichiometry, intensity and stability of interactions. The hybrid technique of ion mobility-mass spectrometry (IM-MS) can provide insight into the conformations adopted by protein and peptide monomers and multimers, in addition to complexes resulting from interactions, which when coupled with molecular modelling can suggest candidate conformations for these in vacuo species and by inference their conformations in solution prior to ionisation and desolvation. The work presented in this thesis considers a number of different peptide and protein systems, highlighting how the combination of MS and IM-MS based techniques, in conjunction with other biophysical techniques such as circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM) and isothermal titration calorimetry (ITC) can provide insight into these dynamic systems. First a case study into the ability of MS and IM-MS to study disorder-to-order transitions is presented. The transcription factor c-MYC can only perform its function upon binding with its binding partner MAX; deregulation of c-MYC is, however, implicated in a number of human cancers. c-MYC and MAX comprise intrinsically disordered regions which form a leucine zipper upon binding. The work presented here focuses on the leucine zipper regions of both c-MYC and MAX, their individual conformations and changes upon binding. Inhibiting the c-MYC:MAX interaction is a current target for drug therapy and hence the inhibition of this interaction with a previously identified small drug-like molecule was also examined using these techniques, to determine if such an approach may be appropriate for investigation of future therapeutics. Next the ability of MS-based techniques to preserve, transmit and distinguish between multiple conformations of a metamorphic protein was examined. The chemokine lymphotactin has been shown to exist in two distinct conformations in equilibrium in a ligand-free state. The existence of such metamorphic proteins has called into question whether traditional structural elucidation tools have been inadvertently biased towards consideration of single conformations. Here, the potential of gas-phase techniques in the study of conformationally dynamic systems is examined through the study of wild type lymphotactin and a number of constructs designed either as a minimum model of fold or to mimic one of the distinct folds. Interactions between chemokines and glycosaminoglycans (GAGs) are thought to be essential for the in vivo activity of these proteins. The interactions between the distinctive chemokine lymphotactin and a model GAG were hence probed. As with the structural studies, additional protein constructs were considered either to represent the minimum model of fold, one distinct fold of the metamorphic protein or designed to diminish its GAG binding propensity. The ability of each construct to bind GAGs, the stoichiometry of the interactions and conformations adopted by the resulting complexes in addition to aggregation occurring upon the introduction of the GAG is considered. Finally, the similarities, with respect to structure and function, between the chemokine superfamily of proteins and the human β-defensin subfamily of antimicrobial peptides are considered. The tendency of human β-defensins 2 and 3 to bind a model GAG is examined; the stoichiometry of binding and conformations adopted and aggregation occurring here are considered and compared with that of chemokines.

Chemical imaging by mass spectrometry is a powerful approach by which to map spatial distributions of molecules to better understand their function in the system of interest. Over the last thirty years, MSI has evolved into a very powerful analytical tool for the investigation of chemically-complex samples including biological tissues, catalytic surfaces and thin layer chromatography plates, among many others. The work in this dissertation aimed to characterize existing MSI methods, while also developing novel instrumentation able to overcome the challenges found in a variety of applications. Different sample preparation and ionization techniques were evaluated to maximize detection of lipid species in brain tissues subjected to traumatic injury to better understand the biological processes involved. Next, differential mobility separation was coupled to an ambient MSI system that resulted in increased signal-to-noise ratios and image contrast. Third, bulky catalytic granite surfaces were imaged to determine specific mineral reactivity and demonstrate the ability of desorption electrospray ionization to image such samples. Fourth, a novel technique was developed names Robotic Plasma Probe Ionization (RoPPI), which uses a vision system-guided robotic arm to probe irregular surfaces for three dimensional surface imaging. Finally, a software program was developed to automatically screen MSI datasets acquired from thin layer chromatography separations for spot-like shapes corresponding to mixture components; this program was named DetectTLC. This research resulted in instrumentation advances for MSI that have enabled increased chemical diversity, enhanced sensitivity and image contrast, imaging of bulky or irregularly-shaped surfaces, and multivariate tools to facilitate data interpretation.

<p>High-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) are powerful tools for the characterization of the molecular structures of components of both simple and complex mixtures. MS and MS/MS have played key roles in many fields, including proteomics, metabolomics, and petroleomics. This thesis focuses on the development of tandem mass spectrometric methods for the structural characterization of asphaltenes and small isomeric molecules. In addition, this thesis also presents a method to address a sampling bias in asphaltene analysis. </p>

High-throughput techniques are currently some of the most promising methods to study molecular biology, with the potential to improve medicine and enable new biological applications. In proteomics, the large scale study of proteins, the leading method is mass spectrometry. At present researchers can routinely identify and quantify thousands of proteins in a single experiment with the technique called shotgun proteomics. A challenge of these experiments is the computational analysis and the interpretation of the mass spectra. A shotgun proteomics experiment easily generates tens of thousands of spectra, each thought to represent a peptide from a protein. Due to the immense biological and technical complexity, however, our computational tools often misinterpret these spectra and derive incorrect peptides. As a consequence, the biological interpretation of the experiment relies heavily on the statistical confidence that we estimate for the identifications. In this thesis, I have included four articles from my research on the accuracy of the statistical confidence estimates in shotgun proteomics, how to accomplish and evaluate it. In the first two papers a new method to use pre-characterized protein samples to evaluate this accuracy is presented. The third paper deals with how to avoid statistical inaccuracies when using machine learning techniques to analyze the data. In the fourth paper, we present a new tool for analyzing shotgun proteomics results, and evaluate the accuracy of  its statistical estimates using the method from the first papers. The work I have included here can facilitate the development of new and accurate computational tools in mass spectrometry-based proteomics. Such tools will help making the interpretation of the spectra and the downstream biological conclusions more reliable.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. EMBARGOED-this thesis is subject to a six months embargo (07/09/06) and may only be viewed and copied with the permission of the author. For further information please Contact the Archivist. Includes bibliographical references: leaf 159-194.

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: David Dickensheets. Includes bibliographical references (leaves 94-95).

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vii, 44 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 41-44).