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.

The ability to characterize the interactions between ions and solvent molecules plays a critical role in understanding fundamental aspects of thermodynamics in solution chemistry. These interactions are often difficult if not impossible to observe in solution due to the number of solvent molecules far exceeding that of the ions. However, this challenge can be circumvented in the gas phase which enables the isolation and study of reactions between a single ion and single solvent molecule. Within the field of ion-molecule chemistry are two sub-categories of interactions known as ionic hydrogen bonds (IHBs) and ionic halogen bonds (IXBs). In these interactions, the incorporation of a charged species permits ion-dipole interactions which are innately stronger than those found in dipole-dipole interactions among neutral molecules. This dissertation describes and explains the interactions which take place between halogenated benzenes (F-, Cl-, Br-, and Iodobenzene) and neutral polar molecules (water, acetonitrile, acetone, and methanol). Additional studies on ionic hydrogen bonding involve the exploration of protonated benzonitrile monomer and dimer solvated by methanol. All systems were examined using the mass-selected ion mobility technique using the VCU mass selected ion mobility mass spectrometer. Thermochemical equilibrium measurements, in conjunction with density functional theory (DFT) calculations, were performed, enabling comparison between experimentally and theoretically determined binding energies. Additionally, the DFT calculations were able to validate hypothetical predictions for the lowest energy structures of each interaction. Furthermore, the averaged collision cross sections of the benzonitrile dimer radical cation, protonated benzonitrile dimer, and benzonitrile solvated hydronium ion were elucidated using the technique of ion mobility where experimentally determined cross sections were compared with theoretical collision cross section calculations on predetermined geometries that were optimized using DFT calculations.

Investigating the structure of proteins and their interactions with other biomolecules or drug molecules, coupled with the consideration of conformational change upon binding, is essential to better understand their functions. Mass spectrometry (MS) is emerging as a powerful tool to study protein and peptide structure and interactions due to the high dynamic range, low sample consumption and high sensitivity of this technique, providing insight into the stoichiometry, intensity and stability of interactions. The hybrid technique of ion mobility-mass spectrometry (IM-MS) can provide insight into the conformations adopted by protein and peptide monomers and multimers, in addition to complexes resulting from interactions, which when coupled with molecular modelling can suggest candidate conformations for these in vacuo species and by inference their conformations in solution prior to ionisation and desolvation. The work presented in this thesis considers a number of different peptide and protein systems, highlighting how the combination of MS and IM-MS based techniques, in conjunction with other biophysical techniques such as circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM) and isothermal titration calorimetry (ITC) can provide insight into these dynamic systems. First a case study into the ability of MS and IM-MS to study disorder-to-order transitions is presented. The transcription factor c-MYC can only perform its function upon binding with its binding partner MAX; deregulation of c-MYC is, however, implicated in a number of human cancers. c-MYC and MAX comprise intrinsically disordered regions which form a leucine zipper upon binding. The work presented here focuses on the leucine zipper regions of both c-MYC and MAX, their individual conformations and changes upon binding. Inhibiting the c-MYC:MAX interaction is a current target for drug therapy and hence the inhibition of this interaction with a previously identified small drug-like molecule was also examined using these techniques, to determine if such an approach may be appropriate for investigation of future therapeutics. Next the ability of MS-based techniques to preserve, transmit and distinguish between multiple conformations of a metamorphic protein was examined. The chemokine lymphotactin has been shown to exist in two distinct conformations in equilibrium in a ligand-free state. The existence of such metamorphic proteins has called into question whether traditional structural elucidation tools have been inadvertently biased towards consideration of single conformations. Here, the potential of gas-phase techniques in the study of conformationally dynamic systems is examined through the study of wild type lymphotactin and a number of constructs designed either as a minimum model of fold or to mimic one of the distinct folds. Interactions between chemokines and glycosaminoglycans (GAGs) are thought to be essential for the in vivo activity of these proteins. The interactions between the distinctive chemokine lymphotactin and a model GAG were hence probed. As with the structural studies, additional protein constructs were considered either to represent the minimum model of fold, one distinct fold of the metamorphic protein or designed to diminish its GAG binding propensity. The ability of each construct to bind GAGs, the stoichiometry of the interactions and conformations adopted by the resulting complexes in addition to aggregation occurring upon the introduction of the GAG is considered. Finally, the similarities, with respect to structure and function, between the chemokine superfamily of proteins and the human β-defensin subfamily of antimicrobial peptides are considered. The tendency of human β-defensins 2 and 3 to bind a model GAG is examined; the stoichiometry of binding and conformations adopted and aggregation occurring here are considered and compared with that of chemokines.

Gas-phase analytical techniques provide very valuable tools for tackling the structural complexity of macromolecular structures such as those encountered in biological systems. Conformational dynamics of polypeptides and polypeptide assemblies underlie most biological functionalities, yet great difficulties arise when investigating such phenomena with the well-established techniques of X-ray crystallography and NMR. In areas such as these ion mobility interfaced with mass spectrometry (IMMS) and molecular modelling can make a significant contribution. During an IMMS experiment analyte ions drift in a chamber filled with an inert gas; measurement of the transport properties of analyte ions under the influence of a weak electric field can lead to determination of the orientationally-averaged collision cross-section of all resolved ionic species. A comparison with cross-sections estimated for model molecular geometries can lead to structural assignments. Thus IMMS can be used effectively to separate gas-phase ions based on their conformation. The drift tube employed in the experiments described herein is thermally regulated, which also enables the determination of collision cross-sections over a range of temperatures, and can provide a view of temperature-dependent conformational dynamics over the experimental (low microsecond) timescale. Studies described herein employ IMMS and a gamut of other MS-based techniques, solution spectroscopy and – importantly – molecular mechanics simulations to assess a) conformational stability of isolated peptide ions, with a focus on small model peptides and proteins, especially the Trp cage miniprotein; and b) structural characteristics of oligomeric aggregates of an amyloidogenic peptide. The results obtained serve to clarify the factors which dominate the intrinsic stability of non-covalent structure in isolated peptides and peptide assemblies. Strong electrostatic interactions are found to play a pivotal role in determining the conformations of isolated proteins. Secondary structures held together by hydrogen bonding, such as helices, are stable in the absence of solvent, however gas-phase protein structures display loss of their hydrophobic cores. The absence of a polar solvent, “self-solvation” is by far the most potent force influencing the gas-phase configuration of these systems. Geometries that are more compact than the folded state observed in solution are routinely detected, indicating the existence of intrinsically stable compact non-native states in globular proteins, illuminating the nature of proteins’ ‘unfolded’ states.

Nanoparticles, simple yet groundbreaking objects have led to the discovery of invaluable information due to their physiological, chemical, and physical properties, have become a hot topic in various fields of study including but not limited to chemistry, biology, and physics. In the work presented here, demonstrations of various applications of chemical free nanoparticles are explored, from the determination of a non-invasive method for the study of the exposome via using soft-landing ion mobility (SLIM) deposited nanoparticles as a matrix-assisted laser desorption/ionization (MALDI-MS) matrix replacement, to the direct SLIM-exposure of nanoparticles onto living organisms. While there is plenty of published work in soft-landing at operating pressures of 1 Torr, the work presented here shows how this technology can be operated at the less common ambient pressure. The ease of construction of this instrument allows for various modifications to be performed for a wide array of applications, furthermore the flexibility in metallic sample, operating pressure, and deposition time only open doors to many other future applications. The work presented will also show that our ambient SLIM system is also able to be operated for toxicological studies, as the operation at ambient pressure opens the door to new applications where vacuum conditions are not desired.

No description available.

Travelling wave ion mobility spectrometry (TWIMS) in combination with ultra-high performance liquid chromatography (UHPLC) and mass spectrometry (MS) has been applied successfully for the untargeted, global metabolic profiling of biofluids such as mouse plasma and saliva. Methods based on UHPLC-MS alone and in combination with ion mobility spectrometry (UHPLC-IM-MS) have been developed and validated for the untargeted metabolite profiling of saliva, obtained non-invasively by passive drool. Three separate metabolic profiling studies have been carried out in conjunction with bioinformatics strategies to identify potential metabolomic biomarker ions that are associated with efficacy of rice bran in colorectal cancer, physiological stress and that have the potential for the diagnosis of asthma. The advantages offered by the utility of ion mobility in UHPLC-MS based metabolic profiling studies, including the increased analytical space, mass spectral clean-up of contaminants such as PEG post-UHPLC-IM-MS analysis, enhancement of the selectivity of targeted metabolites as well as the potential for the identification of metabolites by comparison of ion mobility drift times have been highlighted. Ten potential metabolic biomarker ions of asthma have been identified from the moderate asthmatics from untargeted metabolite profiling of saliva by UHPLC-MS. A predictive model based on partial least squares discriminant analysis (PLS-DA) has been constructed using these ten discriminant ions, which demonstrates good predictive capability for moderate asthmatics and controls. Potential metabolic biomarker ions of physiological stress have been identified through untargeted metabolite profiling analysis of saliva samples collected before and after exercise by UHPLC-IM-MS. Valerolactam has been identified as a potential biomarker of physiological stress from saliva by comparison of retention time, ion mobility drift time and MS/MS spectra with a standard of δ-valerolactam.

The increased use of mass spectrometry in the clinical setting has led to a demand for high sample throughput. Developments such as ultra high performance liquid chromatography and the ambient ionization techniques enable high sample throughput by reducing chromatographic run times or by removing the requirement for sample preparation and fractionation prior to analysis. This thesis assesses the reproducibility and robustness of these high throughput techniques for the analysis of clinical and pharmaceutical samples by ion mobility-mass spectrometry. The rapid quantitative analysis of the urinary biomarkers of chronic obstructive pulmonary disease, desmosine and isodesmosine has been performed by ultra high performance liquid chromatography combined with ion mobility-mass spectrometry. The determination of health status based on the free unbound fraction rather than the total bound and unbound desmosine and isodesmosine, significantly reduces the time taken in sample preparation. The potential for direct analysis of the urinary metabolites from undeveloped TLC plates using a solvent extraction surface sample probe is demonstrated. The use of a solvent gradient for the extraction separates urinary metabolites from salts and other matrix components and allows fractionation of the sample as a result of differential retention on the undeveloped RP-TLC plate. This separation, combined with ion mobility-mass spectrometry provides a rapid ambient ionization method for urinary profiling. The combination of a thermal desorption probe with extractive electrospray ionization has been applied to the direct detection of a known genotoxic impurity from a surrogate active pharmaceutical ingredient. The volatility of the impurity compared to the matrix, allowed selective thermal desorption of the analyte, which was ionized by extractive electrospray and detected by mass spectrometry. The use of a rapid on-probe derivatisation reaction, combined with thermal desorption is demonstrated for the direct determination of urinary creatinine. The aqueous acylation of creatinine significantly increases the volatility of the analyte enabling separation from the urine matrix and analysis by thermal desorption extractive electrospray combined with ion mobility-mass spectrometry.

Structure determination of macromolecular protein assemblies remains a challenge for well-established experimental methods. In this thesis, an emerging structural technique, ion mobility-mass spectrometry (IM-MS) is explored. An assessment of collision cross section (CCS) measurement accuracy using travelling-wave IM (TWIMS) instrumentation was carried out (Chapter 3). Through the collation of a protein complex CCS database and the development of a calibration framework for TWIMS, significant improvements to CCS measurement accuracy have been achieved. Next, the advantages and limitations of using IM-MS to generate restraints for structure characterisation were explored. Computational tools designed to exploit IM-MS data for structural modelling were developed and tested on a training set of systems (Chapter 4). These include two heteromeric protein complexes, and an oligomeric intermediate involved in beta-2-microglobulin aggregation. Further structural information can be attained by using gas-phase dissociation techniques, such as collision-induced dissociation (CID). The effects of charge state on CCS and the gas-phase dissociation pathway of complexes were investigated (Chapter 5). This highlighted the possibility of using CID in conjunction with supercharging to manipulate dissociation pathways to achieve more useful structural information. Finally, the gas-phase structures of globular and intrinsically disordered protein complexes were probed by IM-MS and molecular dynamics (MD) simulations (Chapter 6). Experimental observations were recapitulated remarkably closely by simulations, including gas-phase structural collapse and the ejection of monomer subunits when the energy of the system was increased sufficiently. Overall, this research has contributed to the IM-MS field by providing the framework for improved CCS measurements of large protein complexes and the use of restraints from IM-MS for structural modelling. Significantly, IM-MS has been used in combination with charge manipulation, CID and MD simulations to reveal further insights into the gas-phase structures, stabilities and dissociation pathways of multimeric protein complexes.

Ion mobility (IM) spectrometry is a gas-phase electrophoretic technique in which ions are separated on the basis of their relative mobility in the presence of a weak electric field gradient and a buffer gas. Ion mobility-mass spectrometry (IM-MS) has the capability of separating ions based on m/z, size and shape, providing additional structural information compared to using mass spectrometry on its own. In this thesis, IM-MS has been used to investigate organic and organometallic complexes and identify reactants, intermediates and products in reaction mixtures. Collision cross sections (CCS) have been measured for three salen ligands, and their complexes with copper and zinc using travelling-wave ion mobility-mass spectrometry (TWIMS) and drift tube ion mobility-mass spectrometry (DTIMS), allowing a comparative size evaluation of the ligands and complexes. CCS measurements using TWIMS were determined using peptide and TAAH calibration standards with good intra-day and inter-day reproducibility. TWIMS measurements gave significantly larger CCS than DTIMS derived data in helium, indicating that the choice of calibration standards is important in ensuring the accuracy of TWIMS derived CCS measurements. The CCS data obtained from IM-MS measurements have been compared to CCS values obtained from X-ray coordinates and modelled structures. The analysis of small organic and organometallic molecules has been extended to investigations of the potential of IM-MS for reaction monitoring and structural studies of the components of catalytic cycles. Reaction mixtures of an organocatalysed Diels-Alder cycloaddition reaction have been monitored using IM-MS and high-field asymmetric waveform ion mobility-mass spectrometry (FAIMS-MS). Reactant, product, catalyst and reaction intermediates, including an intermediate not previously detected, were identified and the catalyst and intermediates monitored over time. An organometallic catalytic cycle using a palladium catalyst has been analysed using IM-MS and the CCS of reactants, intermediates and products have been measured and compared to theoretical CCS calculations. Good agreement was observed between measured and calculated data. Species not amenable to electrospray ionisation were covalently bound to an ionisable tag containing a quaternary ammonium ion allowing the tagged molecules to be detected by IM-MS.