Along with genes and proteins, metabolites play important roles in sustaining life. There remains much to be learned about the in vivo roles of metabolites. Metabolomics is a comparative tool to study global metabolite levels in samples under various conditions. This dissertation describes the development and application of metabolomics strategies for discovery of biologically active or novel metabolites with priori knowledge about genes, proteins, or phenotypes. The power of metabolomics for discovery of novel metabolites from genes is demonstrated through the work with the pyochelin (pch) gene cluster. Comparison of the extracellular metabolomes of pch gene cluster mutants to the wild-type Pseudomonas aeruginosa (strain PA14) identified 198 ions regulated by the pch genes. In addition to known metabolites, a pair of novel metabolites were characterized as 2-alkyl-4,5-dihydrothiazole-4-carboxylates (ATCs). Subsequent assays revealed that ATCs bind iron and that their production is regulated by iron levels and dependent on pchE gene in the pch gene cluster. Metabolomics can also facilitate discovery of active metabolites from proteins, as shown in the work with orphan nuclear receptor Nur77. We applied a metabolomics platform for detected protein-metabolite interactions to identify lipids that bind to Nur77. Using this approach, we discovered that the Nur77 ligand-binding domain (Nur77LBD) enriched unsaturated fatty acids (UFAs) in tissue lipid mixtures. Subsequent biophysical and biochemical assays indicate that UFAs bind to Nur77LBD to cause changes in the conformation and oligomerization of the receptor. Last, analogous to classic fractionation experiments, metabolomics can also be applied to discover active metabolites from phenotypes. Using combination of genetics, biochemistry, and metabolomics, we identified three phenazine compounds produced by Pseudomonas aeruginosa that are toxic to the nematode Caenorhabditis elegans. 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of PCA and pyocyanin are strictly pH-dependent at non-overlapping pH ranges. The diversity within a class of metabolites can be used to modulate bacterial toxicity in different environmental niches. / Chemistry and Chemical Biology

Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has become the preferred method for large-scale peptide and phosphopeptide identification and quantification. The dominance of LC-MS/MS is the result of improved chromatographic, mass spectrometry and bioinformatic technologies. The applications of these technological improvements drive biological innovation by expanding the realm of possible experimentation, facilitating the creation and evaluation of novel hypotheses. Such improvements are the focus of this dissertation. New technologies are presented and their proteome wide applications in biological systems are demonstrated. A comparison of common phosphopeptide enrichment methods is presented in chapter two, which demonstrates that a combination of methods provides non-overlapping data sets. This comparison was performed in mitotically arrested fission yeast, a previously unstudied system by phosphoproteomic methods. This chapter remarks upon phosphorylation site conservation between lower and higher eukaryotes, as a means of predicting potentially relevant phosphorylation events in mammals. A new protocol for tissue based peptide quantification is presented in chapter three. The large-scale application of this method is detailed in a system of mouse liver phosphorylation, between fasted and re-fed states. The effect of peptide and protein level false discovery rates on the accuracy of phosphorylation site quantification is highlighted. This method is a cost-effective alternative to available techniques, such as metabolic labeling, and expands the application of proteomics to include larger animals. Finally, an in depth analysis of quantitative LC-MS/MS based multiplexing is the subject of the last chapter. New techniques for peptide pre-fractionation and ion quantification are discussed, which improve proteome coverage and quantitative accuracy. This proteome-wide multiplexing is applied to an analysis of the budding yeast environmental stress response. Applicable methods of data processing and a means of obtaining biologically relevant information out of multidimensional proteomic data sets are discussed. In all chapters, the data presented represent the largest analyses of their kind. This dissertation provides a solid guide for future proteome-wide studies, focused on the identification and quantification of peptides and their posttranslational modifications.

The neurochemical problem (1) and the directive of the neuroanalytical chemist (2) can be stated as follows: (1) The chemical space of the nervous system is populated by hundreds of neuroactive species linked through extensive biological circuits which are dynamically changing in time and space in response to myriad inputs. (2) Neurochemical analysis techniques should therefore have the appropriate temporal, spatial, and chemical resolution to study these systems, while perturbing them so minimally as to allow unfettered in vivo measurements. New tools and concepts for analytical measurements of neurotransmitters, neuropeptides, and synaptic vesicles are developed and presented in this dissertation on analytical measurements for addressing the neurochemical problem. The introduction gives a broad overview of chemical neuroscience and introduces quantitative visualization of the multidimensional resolution paradigm for analytical chemists seeking to design effective experiments. Chapters two through four detail advancements in data processing and instrument design which decrease detection limits and allow for improved spatial, temporal, chemical resolution in capillary electrophoresis measurements of neurotransmitters, metabolites, and synaptic vesicles. Chapter five discusses the development of fast-scan controlled-adsorption voltammetry which has dramatically increases the spatial and temporal resolution of basal dopamine measurement in vivo. Chapter six introduces online-preservation microdialysis as a way to overcome enzymatic degradation of endogenous opioid neuropeptides during in vivo sample collection. Because of this discovery of the secretory behavior these neuropeptides is reported in the anterior cingulate cortex (ACC), a region of the brain deeply associated with pain signaling. The advancement of peptide drugs particularly glycosylated neuropeptide analogs through new methods of mass spectrometry analysis for rapid feedback in drug development are presented in chapter 7. Chapter eight concludes this work with future directions pointing towards single-cell electrochemical and mass spectrometry measurements, shotgun-microdialysis for high-throughput screening of neurotherapeutics, preliminary data on the effect of chronic pain of endogenous opioids in the ACC, and the beginnings of in vivo neuroproteomics analysis in rodent pain models.

No description available.

Background- Severe Plasmodium falciparum malaria remains one of the major causes of childhood morbidity and mortality in Africa. Severe malaria manifests itself as three main clinical syndromes-impaired consciousness (cerebral malaria), respiratory distress and severe malarial anaemia. Cerebral malaria and respiratory distress are major contributors to malaria mortality but their pathophysiology remains unclear. Motivation/Objectives- Most children with severe malaria die within the first 24 hours of admission to a hospital because of their pathophysiological conditions. Thus, along with anti-malarial drugs, various adjuvant therapies such as fluid bolus (for hypovolaemia) and anticonvulsants (for seizures) are given to alleviate the sick child’s condition. But these therapies can sometimes have adverse effects. Hence, a clear understanding of severe malaria pathophysiology is essential for making an informed decision regarding adjuvant therapies. Methodology- We used mass spectrometry-based shotgun proteomics to study plasma samples from Gambian children with severe malaria. We compared the proteomic profiles of different severe malaria syndromes and generated hypotheses regarding the underlying disease mechanisms. Results/Conclusions- The main challenges of studying the severe malaria syndromes using proteomics were the high complexity and variability among the samples. We hypothesized that hepatic injury and nitric oxide play roles in the pathophysiology of cerebral malaria and respiratory distress.

A proteomic procedure for analyzing mouse left ventricular collagen by mass spectrometry has been developed. The procedure involves a purification step that removes non-collagenous cellular components from the collagen extracellular matrix, a step that solubilizes the collagen in aqueous solvents before it is proteolytically digested for analysis with ESI-LCMS/MS. Collagen from healthy and lathrytic mice has been positively identified by applying the SEQUEST database search algorithm to spectra from the collagen prepared using this procedure. Analysis shows that the relative percentage of collagen peptides detected in lathrytic tissue is significantly greater than that of the healthy tissue. These preliminary results suggest that the percentage of cross-linked collagen is lower in the lathrytic tissue as indicated by the greater protein sequence coverage obtained for this tissue. This procedure lays the ground work for future experimentation that has the ability to allow for the identification and quantification of cross-linked peptides.

Burkholderia pseudomallei is the causative agent of Melioidosis, an endemic disease in South East Asia, and is classified as a category B biological agent. Currently, there is no licensed vaccine for this disease; the mortality rate is high due to the incorrect diagnosis and the pathogen insusceptibility to general antibiotics. A mass spectrometry based proteomic approach has been applied in order to identify the proteins that are responsible for pathogenicity.Methods were developed for the proteomic analysis of Burkholderia species using B. vietnamiensis G4, an opportunistic pathogen as the model organism. Both gel-based (LC-MS/MS) and gel-free MudPIT (LC/LC-MS/MS) approaches have been applied for the analysis of the proteins extracted from four different cellular fractions of these bacteria. More than 1200 proteins were identified from these analyses, including many proteins previously identified as virulence factors of these bacteria. Similar methodologies were applied to build a proteome map of non-pathogenic B. thailandensis E264 to use as a reference for the pathogenic studies. Additionally, proteomes of two B. thailandensis strains isolated from two geographical locations were compared to investigate the differences in protein expression of these organisms.Proteins identified from pathogenic B. pseudomallei were compared with the non-pathogenic B. thailandensis and opportunistic pathogen B. vietnamiensis proteins. Many species specific proteins were identified from this proteomic analyses; those proteins can be used as antigen targets to selectively identify these pathogenic bacteria in a complex biological matrix using affinity capture methods.Ticks are vectors that can transmit disease causing pathogens one host to another. Knowing the pathogen reservoir is important in order to control disease spread in the environment. Application of mass spectrometric methods to identify the host blood components from tick vectors was investigated using tick nymphs which feed only once in their life cycle. Using mass spectrometry based proteomics; host specific proteins like hemoglobin and immunoglobulin were identified from a single tick nymph analysis. Additional studies have examined the fatty acid profiles of rabbit and sheep blood fed tick nymphs using SPALDI mass spectrometry. Different fatty acid profiles were obtained for these tick nymphs, but further investigations are required to validate these findings.

Presented in this dissertation are studies of protein dynamics and protein/protein interactions using solution phase hydrogen/deuterium exchange in combination with mass spectrometry (HXMS). In addition, gas phase fragmentation behaviors of deuterated peptides are investigated, with the purpose of increasing resolution of the HXMS. In the area of single protein dynamics, two protein systems are studied. Studies on the cytochrome c2 from Rhodobacter capsulatus indicate its domain stability to be similar to that of the horse heart cytochrome c. Further comparison of the exchange kinetics of the cytochrome c2 in its reduced and oxidized state reveals that the so-called hinge region is destabilized upon oxidation. We also applied a similar approach to investigate the conformational changes of photoactive yellow protein when it is transiently converted from the resting state to the signaling state. The central β-sheet of the protein is shown to be destabilized upon photoisomerization of the double bond in the chromophore. Another equally important question when it comes to understanding how proteins work is the interactions between proteins. To this end, two protein complexes are subjected to studies by solution phase hydrogen deuterium exchange and mass spectrometry. In the case of LexA/RecA interaction, both proteins show decreases in their extents of exchange upon complex formation. The potential binding site in LexA was further mapped to the same region that the protein uses to cleave itself upon interacting with RecA. In the sHSP/MDH system, hydrogen/deuterium exchange experiments revealed regions within sHSP-bound MDH that were significantly protected against exchange under heat denaturing condition, indicative of a partially unfolded state. Hydrogen/deuterium exchange therefore provides a way of probing low resolution protein structure within protein complexes that have a high level of heterogeneity. Finally, the feasibility of increasing resolution of HXMS by gas phase peptide fragmentation is investigated by using a peptide with three prolines near the C-terminus. Our data show that deuterium migration indeed occurs during the collision activated dissociation process. Caution is required when interpreting the MS/MS spectra as a way of pinpointing the exact deuterium distribution within peptides.

Protein phosphorylation is a very common posttranslational modification (PTM), which lately has been found to hold the keyrole in the development of many severe diseases, including cancer. Thereby, phosphoprotein analysis tools, generally based on specific enrichment of the phosphoryl group, have been a hot topic during the last decade. In this thesis, two new TiO2-based on-target enrichment methods are developed and presented together with enlightening fundamental results. Evaluation of the developed methods was performed by the analysis of: custom peptides, β-casein, drinking milk, and the viral protein pIIIa. The results show that: i) by optimizing the enrichment protocol (first method), new phosphorylated peptides can be found and ii) by the addition of a separation step after the enrichment (second method), more multi-phosphorylated peptides, which usually are hard to find, could be detected. The fundamental part, on the other hand, shows that the phosphopeptide adsorption is caused by electrostatic interactions, in general follows the Langmuir model, and the affinity increases with the phosphorylation degree. Here, however, the complexity of the system was also discovered, as the adsorption mechanism was found to be affected by the amino acid sequence of the phosphopeptide.

Protein prefractionation is a popular and effective strategy for improved MS analysis of complex proteome mixtures. A challenge of prefractionation is the even partitioning with high recovery of all components of the mixture, particularly hydrophobic proteins. This thesis assesses various proteome prefractionation platforms, with a goal of comprehensive proteome analysis. A more reliable dataset of 1136 S. cerevisiae transmembrane proteins was computationally generated, and used to assess two gel-based platforms (GeLC/MS and GELFrEE/MS). These platforms were determined to be comparable for proteome analysis. The requirement for high-throughput, automated fractionation demands a gel-free separation workflow. Here, a LC-based workflow was optimized, relying on SDS-assisted yeast extraction, organic solvent protein precipitation, and reversed phase separation in a formic acid/isopropanol solvent system. Though this workflow afforded improvements over conventional LC strategies to proteome fractionation, the gel-based platforms were demonstrated to be superior, in terms of their unbiased separation of hydrophobic vs hydrophilic proteins.