Browsing by Subject "Orbitrap"

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    Automated quantification of 13C labeled peptides
    (2013-08) Goldford, Joshua Elliot
    Metabolic flux analysis (MFA) is a technique used to elucidate intracellular reaction rates (fluxes) in a metabolic network. Intracellular fluxes are determined by providing substrate enriched with stable, heavy isotopic label and subsequently measuring the incorporation of label into metabolic end products. This results in metabolic end products consisting of isomers of discrete mass states, termed isotopomers. The resulting isotopomer distributions (MIDs) for each metabolic end product are then used to infer fluxes. Typically, metabolic end products used for MFA are derivatized protein-bound amino acids. Protein is extracted from the sample and hydrolyzed into constitutive amino acids, resulting in a amino acid pools derived from all cellular protein. Each amino acid pool contains amino acids potentially synthesized from different subcellular compartments, subspecies within a culture, or from different time points within the cell cycle. Thus, fluxes inferred from hydrolyzed total protein lack spatial and temporal resolution. However, if amino acid MIDs were to be measured directly from individual proteins, one could derive the fluxes at the time and place for which that particular protein was synthesized. Therefore, obtaining amino acid MIDs from individual proteins could enable spatial and temporal resolution for metabolic flux analysis. One solution would be to purify individual protein and hydrolyze and measure amino acid MIDs. This approach would require a significant amount of protein, is manually intensive and expensive. A much more viable solution utilizes high-throughput and high-resolution mass spectrometry to quantify and identify peptide MIDs, which can be used to infer constitutive amino acid MIDs. However, there is no well-defined, automated framework for the extraction and quantification of peptide MIDs from raw mass spectra.In the first chapter, the conceptual framework and vocabulary need for mass spectrometry and peptide-based MFA are provided, with a statistical emphasis. Chapter 2 provides a review of proteomics instrumentation for peptide based MFA followed by the algorithmic considerations and potential software solutions available for the extraction of peptide MIDs.Chapter 3 will describe the methods developed for the automated extraction and quantification of isotopically enriched peptides, including parameter optimization of existing methods and description of novel clustering and quantification methods. Chapter 4 describes the validation of the methods using three different sets of labeled peptide MIDs. Chapter 5 provides a brief discussion of method and software improvements for both identification and quantification followed by a brief discussion of future work.
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    New analytical methodologies in the study of auxin biochemistry
    (2014-08) Yu, Peng
    Auxin is the essential plant hormone that regulates many aspects of plant growth and development. Plants typically possess highly complex biochemical networks to regulate the homeostasis of the active hormone, through the regulation of biosynthesis, degradation, transport and conjugation. Biosynthesis, among other processes, has been of particular importance and warranted extensive studies over the decades of auxin research. A number of pathways were proposed and some enzymes potentially involved have been characterized. Stable isotope labeling and turnover studies have proven very useful in these investigative efforts. With the advancement of analytical and computational technologies, it is now feasible to concurrently analyze the turnover patterns of all the precursors in the entire auxin biosynthesis network. I devoted chapter two of the dissertation to establish LC-MS methods to concurrently quantify most of the auxin precursors and deployed different isotopic labeling strategies for turnover studies in Arabidopsis thaliana. Preliminary results showed that indole-3-pyruvate (IPyA) and indole-3-acetaldehyde (IAAld) were among the fastest to be labeled and a key regulatory step existed between IPyA and indole-3-acetic acid (IAA). Chapter three reported the discrepancy and the study of the amount of the total IAA determined by alkaline hydrolysis and that of the summation of all known forms of IAA conjugates in Arabidopsis. My results indicated that chemical artifacts induced by harsh chemical treatments were responsible for a significant portion of the unknown putative IAA conjugates. In chapter four, I described a facile way to directly survey a plant extract for its indole profile, notably IAA conjugates, based on high resolution and accurate mass (HR/AM) liquid chromatography-mass spectrometry (LC-MS). The method was successfully applied to Glycine max, Solanum lycopersicum, Cocos nucifera, and Ginkgo biloba. Together, these investigations and developments have led to an improved understanding of auxin metabolism and now provide useful tools for subsequent studies.