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Mass spectrometry plays a significant role in many proteomics experiments owing to its ability to provide high quality, detailed data on complex samples containing proteins and/or their constituent peptides. As with any technology, the capabilities of mass spectrometers are constantly increasing to provide better resolution, faster data acquisition, and more accurate mass measurements. However, the existence and widespread use of previous-generation instruments is not negligible. While these instruments may not have the capabilities of their modern counterparts they are still able to collect useful experimental data, though their limitations can result in trade-offs between certain parameters such as resolution, sample run-time, and tandem MS experiments. This work describes an alternative method of MS data analysis, dubbed Parallel Isotopic Tag Screening (PITS), which seeks to enable higher throughput and the collection of better quality data on such previous generation instruments.

The focus of this research is to combine both computational as well as experimental methods to study the non-covalent interaction between a selected set of proteins with small molecules. Experimentally, a mass spectrometric technique, recently known as electrospray mass spectrometry together with the computational aspect of this research, in the area of molecular modeling and quantum mechanics would be exercised.Due to the soft ionization nature of this process, electrospray (ES) mass spectrometry (MS) has been employed to study a broad class of large proteins and their non-covalent interaction with small structures, making it possible for the mass of these complexes to _be determined with an error of less than 0.1%. For this research, a set of proteins known as histones and a class of structures known as crown ethers were chosen. The ES technique allows the proteins to be prepared in an acidic medium that protonates the basic amino acids that have been exposed by the solvent. This process leaves the protein with a lot of positive charges thereby making the analysis with a single quadrupole mass spectrometer, Extrel ELQ 400 possible. The non-covalent complexation between the histones and the crown ethers is stabilized by hydrogen bonding therefore the positive charges of the protein remain unchanged. This bonding is made possible by the ability of crown ethers to bind ammonium ion or protonated amino groups. The mass of the protein which is divided by the number of its positive charges can be determined by a new kind of linear plot constructed from the ES data. The ion currents from the electrospray ionization technique is a representation of the non-covalent complexation of the histones and the crown ethers which can be observed in the mass spectra. Other information such as, the binding constants, can be obtained from the mass spectra. / Department of Chemistry

Ionic liquids (IL) are salts which are liquid at low temperatures, typically with melting points under 100 °C. In recent years ILs have been treated as novel solvents and used in a wide variety of applications such as analytical and separation processes, electrochemical devices and chemical syntheses. The properties of many ILs have been extensively studied; these studies have primarily focused on the investigation of key physical properties including viscosity, density and solubility. This thesis presents mass spectrometry (MS) and computational data to investigate the intrinsic interactions between a small number of IL ions and also their interactions with contaminants. MS was used to study gas-phase aggregates of three ILs based on the 1-butyl-3- methylimidazolium (C4mim+) cation. The influence of different ion sources was investigated on C4mimCl. Conventional electrospray ionisation (ESI) and nano-ESI techniques were compared with recently developed sonic-spray ionisation (SSI) and plasma assisted desorption ionisation (PADI). SSI was found to be beneficial to the formation of larger aggregates while PADI was significantly less efficient. Gas-phase structures of the singly charged cationic aggregates of C4mimCl were characterised with the aid of collision induced dissociation (CID) and density functional theory (DFT) calculations. Additionally, CID and DFT gave consistent results for the relative stability of the C4mimCl aggregates, showing a good agreement between experiment and theory. Mixed solutions of C4mimCl with a range of metal chloride salts were used to form aggregates incorporating both IL and metal chlorides. LiCl, NaCl, KCl, CsCl, MgCl2 and ZnCl2 were all combined with C4mimCl. Magic number characteristics were observed for a number of pure IL and mixed aggregates. Many of the mixed species were characterised using MS and DFT calculations. In particular, the relative stabilities were determined and the structures of the aggregates were calculated. It was found that the metal ions would normally act as a core for the aggregates with the stability determined by the metal-chlorine binding strength and the steric hindrance of the aggregates. It was necessary to exploit pseudopotentials as opposed to all-electron basis sets for the larger aggregates and aggregates containing heavy atoms. While water is a very effective contaminant for ILs it was not possible to observe gas-phase IL aggregates incorporating this despite using multiple methods. Additionally the presence of protonated aggregates was likewise not observed throughout the range of experiments. Possible structures where these features would be incorporated were studied with DFT to obtain some insight into their lack of formation.

Proteins and peptides can convert from their native form into insoluble highly ordered fibrillar aggregates, known as amyloid fibrils. The process of fibrillogenesis is implicated in the pathogenic mechanisms of many diseases and, although mature fibrils are well characterised by a plethora of biophysical techniques, the initiation and early steps remain, to date, ambiguous. Mass spectrometry can provide invaluable insights into these early events as it can identify the low populated and transient oligomeric species present in the lag phase by their mass to charge ratio. Recent evidence has shown that oligomers formed early in the aggregation process are cytotoxic and may additionally be central to the progression of diseases associated with amyloid fibril presence. The hybrid technique of ion mobility mass spectrometry can be employed to provide conformational details of monomeric and multimeric species present and elucidate the presence of oligomers which possess coincident mass to charge ratios. Molecular modelling, in conjunction with experimental results, can suggest probable monomeric and oligomeric structural arrangements. In this thesis three aggregating systems are investigated: amyloidogenic transthyretin fragment (105-115), insulin and two Aβ peptides. Initially amyloidogenic endecapeptide transthyretin (105-115) is studied as it has been widely utilised as a model system for investigating amyloid formation due to its small size. Secondly insulin, a key hormone in metabolic processes, is investigated as extensive research has been carried out into its aggregation into amyloid fibrils. The formation of insulin amyloid fibrils rarely occurs in vivo; however localised amyloidosis at the site of injection and the aggregation of pharmaceutical insulin stocks present problems. Thirdly the aggregation of A β peptides Aβ (1-40) and Aβ (1-42) and their interactions with an aggregation inhibitor, RI-OR2, are characterised. A (1-42), although less commonly produced in vivo, is more cytotoxic and has a faster aggregation mechanism than Aβ (1-40). Both Aβ peptides are implicated in the aetiology of Alzheimer’s disease whilst RI-OR2 has been reported to prevent the production of high molecular weight oligomers, with particular suppression of Aβ (1-42) aggregation.

β-defensins are a class of mammalian defence peptides with therapeutic potential because of their ability to kill bacteria and attract host immune cells. In order to realise this potential, it is necessary to understand how the functions of these peptides are related to their structures. This thesis presents biophysical analysis of β- defensins and related peptides in conjunction with biological assays. These studies provide new insights into the structure-activity relationships of β-defensins. Ion mobility-mass spectrometry (IM-MS) is used throughout this thesis to probe the tertiary structure of peptides in vacuo and, by inference, make conclusions about their conformations in solution prior to ionisation. Where appropriate, IM-MS is complemented by other techniques, including high performance liquid chromatography and circular dichroism spectroscopy. First, the importance of a C-terminal cysteine residue within the murine β-defensin Defb14 is investigated. The functional and structural implications of chemically modifying the cysteine residue are examined. Second, the N-terminal region of Defb14 is modified by the substitution and deletion of amino acids. Again, the effects on biological activity and structure are discussed. Finally, the functional and structural overlap of β-defensins with another family of proteins – the chemokines – is considered. The oligomerisation of β-defensins and their interaction with glycosaminoglycans is of particular interest: structural data for human β-defensins 2 and 3 in the absence and presence of polysaccharides are presented.

Peripheral steroids and oxycholesterols are important lipid compounds controlling various functions in the human body. Steroid analysis of biological samples is routinely employed in the clinical environment as an essential source of information on endocrine and metabolic disorders. It has been reported that stress related neurosteroids have been implicated in the development and prognoses of neurodegenerative disorders such as Alzheimer’s disease (AD). These compounds have been identified as possible biomarkers in the diagnosis of AD and other neurodegenerative disorders. Therefore, methods for the simultaneous analysis of steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids) are vital in providing useful and more comprehensive data. Homeostasis of cholesterol in the brain is maintained primarily by metabolism to oxysterols, including oxycholesterols. These oxycholesterols act as a transport form of cholesterol as it readily navigates the blood-brain barrier. Oxycholesterols are generally more bioactive than cholesterol and is of interest in pathophysiology. Moreover, if their production in cells and tissues and/or their introduction with dietary animal fat are excessive, oxycholesterols could indeed contribute to the pathogenesis of various disease processes. The first study in this thesis focuses on a novel supercritical fluid chromatography–tandem mass spectrometry method for targeted analysis of eighteen peripheral steroids. The method is simple and fast. It has sufficient sensitivity for quantification of 18 different steroids in small volume human plasma. Therefore, this novel method can be applied for screening many steroids within 5 minutes providing the possibility to use for routine healthcare practice. The second study involves the quantification of three adrenal steroids in plasma from domesticated White Leghorn (WL) chickens and Red Junglefowl (RJF) birds. The domestication effects on stress induced steroid secretion and adrenal gene expression in chickens are evaluated. The third study focuses on determination of more than ten oxycholesterols in biological samples with a gas chromatography–mass spectrometry method and a supercritical fluid–tandem mass spectrometry method.

Recent advances in technology have led to the determination of numerous notable structures of membrane proteins. While they provide valuable information about the structure of membrane proteins these studies often provide static images with potentially limited dynamics, and structural determination often requires truncation of flexible regions, and often utilizes bacterial homologs given the need for stable, heterologous overexpression. In order to better understand allostery at a molecular level, state-dependent crosslinking studies coupled with multidimensional mass spectrometry (MS) were conducted on glycine receptor (GlyR) stabilized in different allosteric states. Predominant allosteric states were stabilized using wild type or mutated receptor in the presence of selected ligands: resting (no ligand), desensitized (saturating glycine) and open state (non-desensitizing ivermectin (IVM)-gated F207A/A288G GlyR). Photo-crosslinking methodology linked with mass spectrometric analysis was developed on systematically generated single Cys mutations in GlyR with both Cys null and IVM sensitive backgrounds to enable the study of state-dependent structures of GlyR in comparative crosslinking studies. Studies were conducted on A41C and H419C mutants. A41 is shown to be in proximity to the pre-M1 and the M2-M3 loop region crucial for gating. Prior to these studies, very little information on H419 was available as it is located in C-terminal tail of the receptor that is often truncated in structural studies conducted on other related pentameric ligand-gated ion channels. These studies identified specific GlyR crosslinks unique to each conformational state and identified potential motions in the receptor upon gating and desensitization. The defined distance constraints will be used to update our model of human α1-GlyR and provide insight into channel function. Significantly, this methodological approach is amenable to study any allosteric protein and complement other high resolution structural studies in identifying protein dynamics. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

Organic aerosols are ubiquitous to the lower atmosphere and there is growing concern about their impact on climate and human health. These aerosols typically have multicomponent compositions that change over time in part due to oxidation by reactive trace gases, such as ozone. A current challenge to the atmospheric research community is to develop better methods of analysis of these particles. Photoelectron resonance capture ionization aerosol mass spectrometry (PERCI-AMS) is an online mass spectrometric method that has been applied to the analysis of organic aerosols. One of its key advantages is that it employs low energy (~ 0 eV) photoelectrons in the ion forming process, which has been shown to minimize fragmentation in the organic analytes, thus simplifying mass spectral interpretation. This dissertation focuses on the application of PERCI-AMS to the analysis of organic particles. Initial emphasis is placed on the heterogeneous reaction of gas phase ozone with liquid oleic acid particles. Products identified included carboxylic acids, aldehydes, and peroxides including alpha-acyloxyalkyl hydroperoxides polymers. The evidence of peroxidic products suggested the stabilization of carbonyl oxide intermediates (i.e. Criegee intermediates) that are formed during ozonolysis. Subsequent PERCI-AMS experiments investigated the reactivity of the stabilized Criegee intermediates. This included investigating the reaction of Criegee intermediates with unsaturated fatty acids and methyl esters. A novel ketone-forming reaction is described in these systems, suggesting the Criegee intermediates can react at a carbon-carbon double bond. Further PERCI-AMS experiments investigated the oxidative processing of particulate amines including octadecylamine and hexadecylamine. Ozonolysis of these amines resulted in strong NO2 - and NO3 - ion signals that increased with the ozone exposure and suggested a mechanism of progressive oxidation. Additionally, a strong ion signal was detected for NO3 -(HNO3), which is the ion core of the most important ion cluster series in the troposphere, NO3 -(HNO3)n(H2O)m. PERCI-AMS was applied to the analysis of ozonized mixed particles of amines with oleic acid or dioctyl sebacate. In the ozonolysis of the amines with oleic acid, products included imines and amides. The routes to the amides were shown to most likely arise from the reactivity of stabilized Criegee intermediates and/or secondary ozonides with the amines. There was also direct evidence of the formation of a surface barrier in the octadecylamine and oleic acid reaction system, which resulted in the retention of oleic acid at high ozone exposures. These experiments have fostered a better understanding of the analytical capacity of PERCI-AMS in assaying the reactivity of organic aerosols as well as giving a more accurate description of the heterogeneous chemistry of these challenging reaction systems. Suggestions for adaptations to PERCI-AMS and future experiments on