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Call number: LD2668 .T4 1963 H37 / Master of Science

BACKGROUND:Mycoplasma hyopneumoniae causes respiratory disease in swine and contributes to the porcine respiratory disease complex, a major disease problem in the swine industry. The M. hyopneumoniae strain 232 genome is one of the smallest and best annotated microbial genomes, containing only 728 annotated genes and 691 known proteins. Standard protein databases for mass spectrometry only allow for the identification of known and predicted proteins, which if incorrect can limit our understanding of the biological processes at work. Proteogenomic mapping is a methodology which allows the entire 6-frame genome translation of an organism to be used as a mass spectrometry database to help identify unknown proteins as well as correct and confirm existing annotations. This methodology will be employed to perform an in-depth analysis of the M. hyopneumoniae proteome.RESULTS:Proteomic analysis indicates 483 of 691 (70%) known M. hyopneumoniae strain 232 proteins are expressed under the culture conditions given in this study. Furthermore, 171 of 328 (52%) hypothetical proteins have been confirmed. Proteogenomic mapping resulted in the identification of previously unannotated genes gatC and rpmF and 5-prime extensions to genes mhp063, mhp073, and mhp451, all conserved and annotated in other M. hyopneumoniae strains and Mycoplasma species. Gene prediction with Prodigal, a prokaryotic gene predicting program, completely supports the new genomic coordinates calculated using proteogenomic mapping.CONCLUSIONS:Proteogenomic mapping showed that the protein coding genes of the M. hyopneumoniae strain 232 identified in this study are well annotated. Only 1.8% of mapped peptides did not correspond to genes defined by the current genome annotation. This study also illustrates how proteogenomic mapping can be an important tool to help confirm, correct and append known gene models when using a genome sequence as search space for peptide mass spectra. Using a gene prediction program which scans for a wide variety of promoters can help ensure genes are accurately predicted or not missed completely. Furthermore, protein extraction using differential detergent fractionation effectively increases the number of membrane and cytoplasmic proteins identifiable my mass spectrometry.

For proteins the link between their structure and their function is a central tenet of biology. A common approach to understanding protein function is to ‘solve’ its structure and subsequently probe interactions between the protein and its binding partners. The first part of this approach is non-trivial for proteins where localised regions or even their entire structure fail to fold into a three-dimensional structure and yet they possess function. These so called intrinsically or inherently disordered proteins (IDP’s) or intrinsically disordered regions (IDR’s) constitute up to 40% of all expressed proteins. IDPs which have crucial roles in molecular recognition, assembly, protein modification and entropic chain activities, are often dynamic with respect to both conformation and interaction, so in the course of a protein’s ‘lifespan’ it will sample many configurations and bind to several targets. For these proteins, there is a need to develop new methods for structure characterization which exploit their biophysical properties. The solvent free environment of a mass spectrometer is ideally suited to the study of intrinsic interactions and how they contribute to structure. Ion mobility mass spectrometry is uniquely able to observe the range of structures an IDP can occupy, and also the effect of selected binding partners on altering this conformational space. This thesis details the technique of ion mobility mass spectrometry and illustrates its use in assessing the relative disorder of p53 protein. The tumour suppressor p53 is at the hub of a plethora of signalling pathways that maintain the integrity of the human genome and regulate the cell cycle. Deregulation of this protein has a great effect on carcinogenesis as mutated p53 can induce an amplified epigenetic instability of tumour cells, facilitating and accelerating the evolution of the tumour. Herein mass spectrometry provides a compelling, detailed insight into the conformational flexibility of the p53 DNA-binding domain. The plasticity of the p53 DNA-binding domain is reflected in the existence of more than one conformation, independent of any conformational changes prompted by binding. The in vacuo conformational phenotypes exhibited by common cancer-associated mutations are determined and the second-site suppressor mutation from loop L1, H115N, is probed whether it could trigger conformational changes in p53 hotspot cancer mutations. The structural basis of the binding promiscuity of p53 protein is investigated; of particular interest is the molecular interaction of the p53 N-terminus with the oncoprotein murine double minute 2, as well as with the antiapoptotic factor B-cell lymphoma-extralarge.

The apoC-III proteoform containing two sialic acid residues (apoC-III2) has different in vitro effects on lipid metabolism compared with asialylated (apoC-III0) or the most abundant monosialylated (apoC-III1) proteoforms. Cross-sectional and longitudinal associations between plasma apoC-III proteoforms (by mass spectrometric immunoassay) and plasma lipids were tested in two randomized clinical trials: ACT NOW, a study of pioglitazone in subjects with impaired glucose tolerance (n = 531), and RACED (n = 296), a study of intensive glycemic control and atherosclerosis in type 2 diabetes patients. At baseline, higher relative apoC-(I)II2 and apoC-III2/apoC-III1 ratios were associated with lower triglycerides and total cholesterol in both cohorts, and with lower small dense LDL in the RACED. Longitudinally, changes in apoC-III2/apoC-III1 were inversely associated with changes in triglycerides in both cohorts, and with total and small dense LDL in the RACED. apoC-III2/apoC-III1 was also higher in patients treated with PPAR-gamma agonists and was associated with reduced cardiovascular events in the RACED control group. Ex vivo studies of apoC-III complexes with higher apoC-III2/apoC-III1 showed attenuated inhibition of VLDL uptake by HepG2 cells and LPL-mediated lipolysis, providing possible functional explanations for the inverse association between a higher apoC-III2/apoC-III1 and hypertriglyceridemia, proatherogenic plasma lipid profiles, and cardiovascular risk.

Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2007. / This study describes the development of simplified analytical methods for the analysis of trace quantities of selected naturally occurring target compounds in complex biological matrices by stir bar sorptive extraction (SBSE) and gas chromatography/mass spectrometry (GC/MS). SBSE facilitates the direct extraction of organic compounds from aqueous samples by allowing the solutes to partition between the aqueous phase and a glass stir bar that is coated with a layer of polydimethylsiloxane (PDMS). The partitioning of polar compounds into the PDMS coating was enhanced by using different derivatization techniques in combination with SBSE. The derivatization of polar functional groups was performed with ethyl chloroformate, acetic acid anhydride, and O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine directly in the aqueous samples. Headspace derivatization of compounds containing a secondary alcohol group was performed directly on the stir bar coating in the presence of acetic acid anhydride vapors. The derivatized compounds were thermally desorbed (TD) and analyzed on-line by GC/MS. A number of experimental parameters, including salt addition, temperature and time were optimized to improve the recovery of the derivatized compounds by SBSE. The optimized methods were validated in terms of linearity, precision, and detection and quantitation limits prior to performing the quantification. Trace levels of tuberculostearic acid, a marker of tuberculosis, was detected in sputum samples that were decontaminated and concentrated before being analyzed by SBSE-TD-GC/MS. The method is sufficiently sensitive to detect the marker without the need to culture the organisms, namely M. Tuberculosis. The analysis of 4-hydroxynonenal has also been demonstrated by detecting trace levels of this oxidative stress marker in urine samples obtained from healthy volunteers. Furthermore, abnormally low testosterone/epitestosterone ratios were detected in a group of HIV positive patients by means of SBSE-TD-GC/MS. Further research is required to determine the clinical significance of this finding in the context of HIV infection. Finally, the excessive urinary excretion of estrone and 17β-estradiol following the administration of a high dose of the conjugated equine estrogens to a female volunteer has also been demonstrated.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Phenolic chemistry is quite complex; natural phenolic compounds vary widely in terms of size and chemical properties. The high structural diversity within this family presents severe analytical challenges. High performance liquid chromatography (HPLC) is the preferred method for phenolic analysis; however, conventional HPLC methods offer limited separation power and often provide incomplete separation of the large number of components present in natural phenolic extracts. Multi-dimensional chromatographic techniques have proven much more effective in the analysis of complex samples. The current study explored the potential of comprehensive two-dimensional liquid chromatography (LC×LC) for the characterisation of phenolic compounds in complex natural products, with the emphasis on proanthocyanidins (PACs). Initial work focused on the evaluation of the state of the art in phenolic analysis, to allow information which was used in the development of optimal 1-D separations for use in LC×LC. The combination of hydrophilic interaction chromatography (HILIC) in the first dimension with reversedphase liquid chromatography (RP-LC) in the second dimension afforded an orthogonal and powerful separation system for phenolics, providing separation on the basis of hydrophilicity and hydrophobicity, respectively. A detailed and systematic procedure was therefore developed to allow the optimisation and evaluation of on-line, off-line and stop-flow HILIC×RP-LC methods. Results showed that all three approaches provide much better separation performance than conventional one-dimensional LC (1-D LC) techniques. On-line HILIC×RP-LC offers automation, shorter analysis times, better reproducibility and minimal sample exposure. The off-line and stopflow methods are characterised by much higher peak capacities, but relatively long analysis times. It was also demonstrated that stop-flow operation results in negligible additional band broadening for procyanidins (PCs), implying that this method is an attractive alternative to the off-line method as it offers automation and minimal sample handling. Experimental verification of the predictions based on fundamental principles confirmed the validity of the optimisation procedure for cocoa PCs. The hyphenation of on-line HILIC×RP-LC separation with fluorescence (FL) and mass spectrometry (MS) detection methods provided enhanced resolution in a practical analysis time with the added benefit of selective detection and greater certainty in compound identification. This strategy proved much more powerful, as demonstrated by the identification of the highly complex PACs in grape seeds based on chromatographic retention data in two dimensions and accurate mass information. It was further shown that on-line coupling of HILIC×RP-LC separation with an optimised radical scavenging assay provides an improved approach for screening of individual radical scavengers in complex phenolic fractions, as demonstrated for cocoa, grape seed and green tea extracts. / AFRIKAANSE OPSOMMING: Fenoliese chemie is baie kompleks; natuurlike fenoliese verbindings varieer in terme van beide grootte en chemiese eienskappe. Hierdie hoë strukturele diversiteit binne die familie bied daadwerklike analitiese uitdagings. Hoëverrigtingvloeistofchromatografie (HPLC) is die voorkeurmetode vir fenoliese analises, maar konvensionele HPLC metodes bied egter 'n beperkte skeidingsvermoë en verskaf dikwels onvolledige skeiding van die groot aantal komponente teenwoordig in natuurlike fenoliese ekstrakte. Multi-dimensionele chromatografiese tegnieke is bewys om baie meer effektief te wees met betrekking tot die ontleding van komplekse monsters. Hierdie studie ondersoek die potensiaal van omvattende twee-dimensionele vloeistof chromatografie (LC×LC) vir die karakterisering van fenoliese verbindings in komplekse natuurlike produkte, met die fokus op pro-antosianidiëne (PAC’s). Aanvanklike werk het gefokus op die evaluering van moderne tegnieke vir fenoliese analise – inligting wat in die ontwikkeling van optimale 1-D skeidings vir die toepasing in LC×LC gebruik is. Die kombinasie van hidrofiliese interaksie chromatografie (HILIC) in die eerste dimensie met omgekeerde-fase vloeistof chromatografie (RP-LC) in die tweede dimensie verleen 'n ortogonale en kragtige skeidingsisteem vir fenoliese komponente en verskaf skeiding op grond van onderskiedelik hidrofiliteit en hidrofobiteit. ‘n Gedetailleerde en sistematiese prosedure is dus ontwikkel om die optimisering en evaluering van aan-lyn, af-lyn en stop-vloei HILIC×RP-LC metodes uit te voer. Resultate het getoon dat al drie benaderings baie beter skeidingsvermoë bied as konvensionele een-dimensionele LC (1-D LC) tegnieke. Aan-lyn HILIC×RP-LC bied outomatisering, korter ontledingstyd, beter herhaalbaarheid en minimale monster blootstelling. Die af-lyn en stop-vloei metodes word gekenmerk deur 'n veel hoër piekkapasiteit, maar relatief lang ontledingstye. Daar is ook getoon dat die stop-vloei prosedure geringe bykomende bandverbreding vir prosianodiniëne (PC’s) tot gevolg het, wat beteken dat hierdie metode 'n aantreklike alternatief is vir die af-lyn metode aangesien dit outomatisering bied en minimale monster hantering behels. Eksperimentele verifiëring van die voorspellings gebaseer op fundamentele beginsels bevestig die geldigheid van die optimalisering proses vir kakao PCs. Die koppeling van aan-lyn HILIC×RP-LC skeiding met fluoressensie (FL) en massaspektrometrie (MS) deteksie verskaf verbeterde resolusie binne 'n praktiese ontledingstyd saam met die bykomende voordeel van selektiewe opsporing en groter sekerheid betreffende die verbindings se identifikasie. Hierdie strategie was baie meer kragtig, soos gedemonstreer deur die identifisering van die hoogs komplekse PAC’s in druiwepitte gebaseer op chromatografiese behoud van die integriteit van die data in twee dimensies tesame met akkurate massa inligting. Daar is verder getoon dat aanlyn koppeling van HILIC×RP-LC skeiding met 'n geoptimiseerde radikale vangers deteksie-metode 'n beter benadering bied om die gedrag van individuele radikale vangers in komplekse fenoliese fraksies te bestudeer, soos bewys is vir kakao, druiwepitte en groen-tee ekstrakte.

When treating patients with cancer, the ability to predict a patient’s response to treatment is an important tool to allow therapy to be tailored for best outcome. Therefore, the need exists for a test to forecast a patient’s response using a sample that is readily accessible, and provides an accurate reflection of a patient’s response to a disease or treatment. Profiling biological fluids, such as plasma or urine, has gained considerable interest in recent years. This is because these fluids are readily available and are expected to provide an accurate representation of a patient’s response to treatment. As such, much effort has been put into finding biomarkers or prognostic indicators. Abnormal protease activity has been linked to the progression of cancer due to their involvement in several processes vital to the survival and proliferation of the disease. These include metastasis, resistance to apoptosis and angiogenesis, amongst others. In addition, dysregulated protease activity has been linked to poor response to chemotherapy as well as tumour regrowth following radiotherapy. Therefore, an increased understanding regarding the activity of a patient’s proteases may provide the clinician with more information as to how best to treat the patient. Therefore, monitoring protease activity has been suggested as a potential marker to predict a patient’s response to cancer treatment. Most enzyme activity assays are currently performed by fluorescence spectroscopy. However, these workflows suffer from limited sensitivity and linear range. Therefore, an alternative, more sensitive assay is required. Mass spectrometry (MS) is a highly sensitive analytical technique routinely used to quantify changes in biological systems. As such, MS has the potential to be used in enzyme activity assays. This study illustrates the development of a novel MS based method to monitor the activity of target enzymes in plasma; specifically asparaginyl endopeptidase (AEP) and caspase-3 using mass spectrometry. These enzymes have been linked to poor chemotherapeutic response in childhood leukaemia and tumour regrowth post-radiotherapy respectively. This project will describe the development and optimisation of each stage of a five step sample preparation and analysis method. This includes the design of enzyme substrates designed to be cleaved by the target enzyme, whilst reducing the effect of other enzymes acting on this substrate, how best to enrich samples for these target peptides, as well as determining the best MS technique to monitor these peptides. In addition, this project describes a comparison between this assay and an existing fluorescence assay when monitoring AEP activity in biological samples such as plasma and whole cell lysates. The application of this method in quantifying caspase-3 activity in plasma samples is also examined.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy