The field of proteomics encompasses the study of identities, interactions, and dynamics of all proteins expressed by a living system. Research in this dissertation blends biochemical and quantitative proteomics techniques to increase the latitude of biological applications for the bottom-up mass spectrometry proteomics approach. Together, isolation of selected protein “targets,” such as multiprotein complexes, and quantitative characterization yields information essential for more detailed understanding of microbial cell function. Often, a challenging aspect of characterizing a variety of biochemically enriched samples is limited protein yield. This dissertation describes an enzymatic proteolysis protocol employing an organic/aqueous solvent that alleviates excessive handling steps to reduce losses during sample preparation for small quantities of protein samples. Presence of artifactual, non-specific proteins in enriched protein complex isolates complicates biological interpretation of specific protein interactions. Heterologous expression of affinity-tagged bait proteins may also cause unintended collateral effects. A series of local and global protein isotope ratio measurements were performed to differentiate authentic interactions from artifactual interactions among affinity-isolated complexes and assess collateral effects, respectively. Protein localization provides clues regarding protein function. To infer protein localization, quantitative proteomics techniques were used to estimate protein enrichment of cold osmotic shock periplasmic isolates. Protein isotope ratios indicating enrichment, combined with identification of amino-terminal signal peptide cleavages, increase confidence of periplasmic localization. Collectively, this dissertation provides a framework for tailoring biochemical and quantitative techniques for targeted characterization of microbial protein isolates.

Introduction: Chronic wounds are a great burden to care-givers and patients alike and are the main cause of many preventable amputations. Such wounds are treated with wound dressings but providing a wound environment that is conducive to proper wound healing is not always possible with such dressings. Absorbatox™ is a natural zeolite that has been manipulated to increase its cationic exchange capacity and has its main functionality as a potential wound healing agent in its strong capillary action. This quality enables the zeolite to absorb excess wound exudate and thus prevent wound infection and maceration. Absorbatox™ was characterised to determine its effects on wound healing. Methods: The physical characterisation of two grades of Absorbatox™ - granular and micronised - was conducted using nitrogen adsorption to determine pore size and surface area, and laser particle sizing to determine the particle sizes of the Absorbatox™ particles. Full-thickness wounds of 8 x 8 mm were created on the backs of pigs and treated with Absorbatox™, a positive and a negative control. The wound dimensions were measured and recorded. The wounds were then excised on selected days of each phase of wound healing and fixed in formalin. The wound sections were analysed by mass spectrometry imaging and abundant wound proteins were identified from the tryptic digests using BLAST against the Swiss-Prot database. Results: The surface areas of the micronised and granular Absorbatox™ were 14.43 and 11.23 m2/g, respectively. The micronised Absorbatox™ particle sizes ranged between 0.8 µm to approximately 300 µm with an average pore diameter of 28.2 nm. The granular Absorbatox™ particle sizes ranged between 2 µm and 875 µm with average pore diameters of 43.8 nm. Absorbatox™ showed better wound healing by delaying wound contraction and causing more rapid shallowing of the wound depths compared to the negative control. The difference observed in the wound healing rates of the Absorbatox™-treated and positive control groups were statistically significant and the histological evaluations of the wounds treated with Absorbatox™ showed wound closures that were associated with qualities that more closely resembled normal, healthy tissue than the positive control wounds. The protein activity in the trypsin-digested tissue including within the wound area and the surrounding healthy tissue was successfully imaged using MALDI-MSI. BLAST software was used at an e-value of 30 to identify possible proteins from the tryptic digests and were identified as proteins involved in wound healing. Discussion: Micronised Absorbatox™ treated wounds showed more rapid healing than the other treatments most likely due to the smaller particles and pores which results in strong capillary action to absorb excess exudate. Mass spectrometry imaging allowed monitoring of the protein fluctuations that occur during wound healing. The proteins detected were then identified using BLAST and MASCOT database comparison tools which identified that the abundant proteins detected by mass spectrometry were not those typically observed in wound healing but rather those involved in molecular aspects of wound healing like nerve regeneration, cell proliferation, survival, and migration. / Dissertation (MSc)--University of Pretoria / Pharmacology / unrestricted

This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....