The design and fabrication of a novel soft landing instrument Soft Landing Ion Mobility (SLIM) is described here. Topics covered include history of soft landing, gas phase mobility theory, the design and fabrication of SLIM, as well as applications pertaining to soft landing. Principle applications devised for this instrument involved the gas phase separation and selection of an ionized component from a multicomponent gas phase mixture as combing technique to optimize coatings, catalyst, and a variety of alternative application in the sciences.

Preparative mass spectrometry is a gas-phase ion deposition technique aimed at deposition of monodisperse ion beams on a surface. This is accomplished through the implementation of a soft-landing ion mobility system which allows for high ion flux of conformationally selected ion packets. The soft-landing ion mobility system has been applied to a number of unique chemical problems including the deposition of insulators on graphene, the preparation of reusable surface enhanced Raman spectroscopic substrates, and the deposition of uranium nanoparticles. Soft-landing ion mobility provided a platform for the quick deposition of usable amounts of materials, which is the major objective of preparative mass spectrometry. Soft-landing ion mobility is unique when compared to other preparative mass spectrometric techniques in that the ion packets are conformationally separated, not separated on mass to charge ratio. This provides orthogonal complementary data to traditional mass spectrometric techniques and allows for the study of conformationally monodisperse surfaces. The diversity of problems that have been and continued to be explored with soft-landing ion mobility highlight the utility of the technique as a novel tool for the study of multiple ion/surface interactions.

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Analysis and characterisation of biomolecules using mass spectrometry has advanced over the past decade due to improvements in instrument design and capability; relevant use of complementary techniques; and available experimental and in silico data for comparison with cutting-edge research. This thesis presents ion mobility data, collected on an in-house modified QToF mass spectrometer (the MoQTOF), for a number of protein systems. Two haemoproteins, cytochrome c and haemoglobin, have been characterised and rotationally-averaged collision cross-sections for a number of multimeric species are presented. Intact multiply-charged multimers of the form [xCyt c + nH]z+ where x = 1 (monomer), x = 2 (dimer) and x = 3 (trimer) for cytochrome c have been elucidated and for species with x ≥ 2, reported for the first time. Fragment ions possibly attributed to a novel fragmentation mechanism, native electron capture dissociation, are reported with a brief discussion into their possible production from the dissociation of the gas-phase dimer species. Haemoglobin monomer globin subunits, dimers and intact tetramer have been successfully transferred to the gas phase, and their cross-sections elucidated. Comparisons with in silico computational data have been made and a discussion of the biologically-active tetramer association/dissociation technique is presented. Three further proteins have been studied and their gas-phase collision cross-sections calculated. Two regions of the large Factor H (fH) complement glycoprotein, fH 10-15 and fH 19-20, have been characterised for the first time by ion mobility-mass spectrometry. Much work using nuclear magnetic resonance spectroscopy has previously been achieved to produce structural information of these protein regions, however further biophysical characterisation using mass spectrometry may aid in greater understanding of the interactions these two specific regions have with other biomolecules. The DNA-binding core domain of the tumour suppressor p53 has been characterised and cross-sections produced in the presence and absence of the zinc metal ion that may control the domain’s biological activity. Within this core domain, p53 inactivation mutations have been shown to occur in up to 50% of human cancers, therefore the potential exists to further cancer-fighting activity through research on this region. Anterior Gradient-2 (AGR2) protein facilitates downregulation of p53 in an as yet unclear mechanism. Recent work using peptide aptamers has demonstrated that this downregulation can be disrupted and levels of p53 restored. Collision cross-sections for six peptide aptamers have been calculated, as well as cross-sections for multimers of AGR2 protein. A complex between one aptamer with the protein has also been elucidated. Use of the commercially available Synapt HDMS ion mobility-mass spectrometer at Waters MS Technologies Centre (Manchester, UK) allowed data to be collected for both Factor H protein regions and for the DNA-binding core domain of p53. Data are compared in the appropriate chapters with data collected using the MoQTOF.

Drift tube ion mobility spectrometry (IMS) is a powerful, post-ionization separation that yields structural information of ions through an ion-neutral collision cross section. The ion-neutral collision cross section is governed by the collision frequency of the ion with the neutral drift gas. Consequently, ions of different size will have different collision frequencies with the gas and be separated in the drift cell. A significant challenge for IMS, however, is to separate ions with very similar collision cross sections, requiring higher resolution ion mobility spectrometers. Resolution in IMS is of utmost importance for the separation of complex mixtures, e.g. crude oil samples, proteolytic digests, positional isomers, and ion conformers. However, most methods employed to increase mobility resolution significantly decrease ion transmission through the mobility device. Herein, a periodic-focusing DC ion guide drift cell (PDC IG) is presented to display its potential capabilities for higher mobility resolution with increased ion transmission. The PDC IG utilizes unique electrode geometry compared to the conventional uniform field electrode design. Electrode geometry can be defined by the electrode inner diameter (d), thickness (t), and spacing (s). Specifically, the ratio of d : t : s is equal to, or very near, 1:1:1. The PDC IG electrode design creates a non-uniform (fringing) electric field-especially near the electrode walls. The design also causes variations in the radial electric field which provides an effective RF as ions move through the device and a radially confining effective potential that improves ion transmission through the device. In this dissertation the analytical utility of the PDC IG drift cell for ion mobility separations will be explored. The radial focusing properties of the device will be presented along with studies of electrode geometry and its effect on ion mobility resolution and ion transmission through the drift cell. PDC IG drift cell length is also examined to determine its effect on mobility resolution and ion transmission. Finally, the PDC IG drift cell device is coupled to an orthogonal-acceleration time-of-flight mass spectrometer as well as a modular, PDC IG drift cell being adapted to a commercial qTOF mass spectrometer for IM-MS experiments.

Drift time ion mobility spectrometry (DTIMS) is a rapid post ionization gas-phase separation technique that distinguishes between compounds based on their differences in reduced mass, charge and collisional cross-section while under a weak, time-invariant electric field. Standalone DTIMS is currently employed throughout the world for the detection of explosives, drugs and chemical-warfare agents. The coupling of IMS to MS (IM-MS) has enabled the performance of time-nested multidimensional separations with high sample throughput and enhanced peak capacity, allowing for the separation of ions not only based on their mass/charge (m/z) ratios, but also their shape. This allows for the elucidation of valuable structural information that can be utilized for determining gas phase ion conformation and differentiation between closely related ionic species. Over the past decade, these advances have transformed IM-MS applications and instrumental designs into one of the most rapidly growing areas of mass spectrometry. The work presented in this thesis is aimed at the development and subsequent characterization of a novel high-resolution resistive-glass atmospheric pressure DTIMS, and the application of this prototype DTIMS to the detection of environmentally relevant compounds. A review of the different types of ion mobility spectrometers, their principles of operation, and the advantages and disadvantages of each type are presented in Chapter 1. Chapter 2 describes the design and development of our prototype resistive glass DTIMS. A detailed description of the IMS hardware, including the ion sources, custom-built control computer, pulsing electronics, data acquisition system, and the timing schemes developed to operate the instrument in standalone DTIMS, multiplexed DTIMS, and IM-MS mode, are presented. Chapter 3 presents an initial characterization of the performance of a prototype resistive glass DTIMS under a wide range of instrumental parameters and also characterizes the radial ion distribution of the ions in the drift region of the spectrometer. Chapter 4 addresses the lack of sensitivity in DTIMS and explores ion trapping and multiplexing methods, introduces the principles of multiplexing and describes an extended multiplexing approach that encompasses arbitrary binary ion injection waveforms with variable duty cycles. Chapter 5 presents a detailed theoretical and experimental study of the separation power of our DTIMS and presents an evaluation of the field homogeneity and the performance of the ion gate.

Preparative mass spectrometry is an important method for the synthesis of new materials. Recently, soft landing mass spectrometry has been used to land ions on surfaces to coat or otherwise alter them. Commercial soft landing instruments do not yet exist, and the physical phenomenon of soft landing has not yet been fully described. For future ion mobility soft landing research, the theory of ion mobility, ion optics and soft landing is discussed, and 2 soft landing instruments have been built and described, along with proof of concept experiments for both instruments. Simulations of the process of ion mobility and ion optics for use in these instruments, as well as some preliminary results for the optics are included. Surfaces described include copper on mica and iron on silicon. Self assembly of soft landed ions is observed on the surfaces. The instruments constructed will be useful for future soft landing research, and soft landing can be used for future materials research with special attention focused on the self-assembly of the landed ions.

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.

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.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy