SgrAI is a restriction endonuclease (ENase) that cuts a long recognition sequence and exhibits self-modulation of cleavage activity and sequence specificity. Previous research has shown that SgrAI forms large oligomers when bound to particular DNA sequences and under the same conditions where SgrAI exhibits accelerated DNA cleavage kinetics. However, the detailed structure and stoichiometry of SgrAI:DNA as well as the basic building block of the oligomers, has not been fully characterized. Ion mobility mass spectrometry (IM-MS) was employed to analyze SgrAI/DNA complexes and show that the basic building block of the oligomers is the DNA-bound SgrAI dimer (DBD). The oligomers are heterogeneous containing a mixture of species with variable numbers of DBD. The collision cross sections (CCS) of the oligomers were found to have a linear relationship with the number of DBD. Models of the SgrAI/DNA oligomers were constructed and a head-to-tail arrangement was most consistent with the experimental CCS.

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.

In this thesis, the performance of a miniaturised field asymmetric waveform ion mobility spectrometry (FAIMS) device hyphenated with time-of-flight mass spectrometry is studied and evaluated for analysis of a variety of compounds in different sample matrices. FAIMS is a selective spectrometer which is highly orthogonal to mass spectrometry and has the potential for enhancing sensitivity and improve selectivity of rapid analyses. In Chapter 2, the performance of the miniaturised FAIMS device is tested for stability and transmission under a wide range of ion source conditions. An investigation of three different systems, including pairs of isobaric, isomeric and near-mass ions shows that miniaturised FAIMS has the ability to distinguish between analytes that are challenging to separate by mass spectrometry. Chapter 3 explores the effect of changing the composition of the carrier gas by observing the effect of adding gas modifiers on the FAIMS spectra of small molecules, peptides and proteins. Chapter 4 investigates the advantages of combining a fast FAIMS separation with mass spectrometry in the analysis of nitrogen-containing pharmaceutical impurities, where FAIMS is found to offer additional selectivity. In Chapter 5, the development of a UHPLC-FAIMS-MS method for the quantitative determination of a drug metabolite in urine is reported. UHPLC-FAIMS-MS shows improvements in signal-to noise and linear dynamic range as well as a reduction in chemical noise, demonstrating the potential of combining FAIMS with mass spectrometry.

Glycans and their conjugates form the largest and most diverse class of biological molecules found within nature. These glycosides are vital for numerous cellular functions including recognition events, protein stabilisation and energy storage, to name a few. Additionally, abnormalities within these structures are associated with a wide range of disease states. As a result, robust analytical techniques capable of in depth characterisation of carbohydrates and their binding partners are required. Currently, liquid chromatography coupled with tandem mass spectrometry (MS2) is the 'gold standard' for characterising these species. However there are inherent challenges for 'sequencing' carbohydrates given that most structures are diastereomeric. As a result MS alone is insufficient to fully elucidate all stereochemical and often regiochemical information and alternative analytical techniques have inherent issues meaning that they are not suitable for medium/high throughput analysis. To facilitate elucidation of these structures, ion mobility spectrometry (IMS) has been used in-line with MS2. IMS of mono- and di-saccharide product ions generate by collision-induced dissociation (CID) of various glycans and their conjugates enables unambiguous identification of the monomer and the regio-/stereo-chemistry of the glycosidic bond, independent of the precursor structure. Also, given the prominence of glycans in biological recognition events, high-throughput techniques capable of elucidating and characterising carbohydrate to glycan-binding protein (GBP) interactions are highly sought after. Historically, (micro)array strategies are employed to screen large numbers of biological interactions, with detection conventionally achieved with fluorescent tagging. The major disadvantage of this approach is the requirement of a labelling step to facilitate detection of glycan-GBP binding. MS offers the ability to unambiguously identify GBPs when combined with routine bottom-up proteomics strategies, namely on-chip proteolysis followed by mass fingerprinting and MS2 analysis and subsequent comparison to protein databases. It is anticipated that these methodologies developed throughout these studies, both for carbohydrate sequencing and the characterisation of glycan-binding proteins, will greatly add to the Glycomics toolbox.

Research into the relationship between the structure and function of proteins has been ongoing now for several decades. More recently, there has been an explosion in the investigation of the dynamic properties of proteins, and how their dynamic propensity relates to their function. This new direction in protein research requires new techniques to analyse protein dynamics, since most traditional techniques are biased towards a fixed tertiary structure. Mass spectrometry (MS) is emerging as a powerful tool to probe protein dynamics since it can provide information on interconverting conformations and has no preference towards the folded state. Furthermore, its low sample consumption, rapid data acquisition and low data processing positions MS as an attractive tool in protein structure research. The hybrid technique of ion mobility-mass spectrometry provides further insight into the range of conformations adopted by proteins and protein complexes, by providing information on the size in terms of rotationally averaged collision cross section. The work presented in this thesis considers proteins with a range of structural characteristics. We use ion mobility mass spectrometry to investigate proteins of different extents of disorder, protein complexes with dynamic entities and a system that undergoes structural rearrangement upon ligand binding. First, a framework of mass spectrometry experiments is described which allows identification of the extent of structure and disorder within proteins. This framework is tested on a range of different systems throughout the thesis. Differences in the gas-phase properties of two conformationally dynamic proteins which behave similarly in solution are investigated and from this research we postulate a new ionisation mechanism for partially folded proteins. The dynamic propensity of C-terminal p27 is investigated and compared to two permutants which allows us to delineate how the location of charged residues in a primary sequence affects the structure of a protein. We monitor the 'folding-upon-binding' behaviour of p27 upon association with its binding partners, and how this differs with the order of charged residues in the linear sequence. Finally, we describe the structural rearrangement of Fdc1 upon the binding of its cofactor; a prenylated FMN molecule. This thesis demonstrates the suitability of ion mobility-mass spectrometry for the investigation of dynamic properties of proteins and protein complexes.

Triacetone triperoxide has been synthesised for a range of experimental studies towards the reactivity, stability and gas phase metal complexation of the explosive. Reaction with transition metals was performed and a series of molten TATP experiments conducted at 120 °C. The effect of a molten state on the degradation, conformation and reactivity of TATP has been studied with the formation of diacetone diperoxide, acetone and acetic acid degradation products observed. The complexation of TATP with a range of metals has been studied by electrospray ionisation-mass spectrometry, yielding [M+Cat]+ ions for all of the alkali metals. The first observations of a [TATP+Ag]+ adduct have been made. The formation of [2TATP+Li+LiX]+ (X = Br, Cl) and [2TATP+Ag]+ sandwich complexes was also observed. Collision cross sections (CCS) for the lithium-containing complexes of TATP were measured by travelling wave ion mobility-mass spectrometry, comparing well to computationally determined structures.

The characterization of biomolecules and biomolecular complexes represents an area of significant research activity because of the link between structure and function. Drug development relies on structural information in order to target certain domains. Many traditional biochemical techniques, however, are limited by their ability to characterize only certain stable forms of a molecule. As a result, multidimensional approaches, such as ion mobility mass spectrometry coupled to mass spectrometry (IMS-MS), are becoming very attractive tools as they provide fast separation, detection and identification of molecules, in addition to providing three-dimensional shape for structural elucidation. The present work expands the use and application of trapped ion mobility spectrometry-coupled to mass spectrometry (TIMS-MS) by analyzing a range of biomolecules (including proteoforms, intrinsically disordered peptides, DNA and molecular complexes). The aim is to i) evaluate the TIMS platform measuring sensitivity, selectivity, and separation of targeted compounds, ii) pioneer new applications of TIMS for a more efficient and higher throughput methodologies for identification and characterization of biomolecular ions, and iii) characterize the dynamics of selected biomolecules for insight into the folding pathways and the intra-or intermolecular interactions that define their conformational space.

The drift time spectra of polycyclic aromatic hydrocarbons (PAH), alkylbenzenes and alkylphenylethers were recorded with a laser-based ion mobility (IM) spectrometer. The ion mobilities of all compounds were determined in helium as drift gas. This allows the calculation of the diffusion cross sections (Omegacalc) on the basis of the exact hard sphere scattering model (EHSSM) and their comparison with the experimentally determined diffusion cross sections (Omegaexp). These Omegaexp/Omegacalc-correlations are presented for molecules with a rigid structure like PAH and prove the reliability of the theoretical model and experimental method. The increase of the selectivity of IM spectrometry is demonstrated using resonance enhanced multiphoton ionisation (REMPI) at atmospheric pressure, realized by tuneable lasers. The REMPI spectra of nine alkylbenzenes and alkylphenylethers are investigated. On the basis of these spectra, the complete qualitative distinction of eight compounds in a mixture is shown. These experiments are extended to alkylbenzene isomer mixtures.

The construction of a liquid nitrogen-cooled ion mobility spectrometer coupled with time-of-flight mass spectrometry was implemented to demonstrate the ability to discriminate between electronic isomers. Ion mobility allows for the separation of ions based on differing cross-sections-to-charge ratio. This allows for the possible discrimination of species with same mass if the ions differ by cross-section. Time-offlight mass spectrometry was added to mass identify the separated peak for proper identification. A liquid nitrogen-cooled mobility cell was employed for a two-fold purpose. First, the low temperatures increase the peak resolution to aid in resolving the separated ions. This is necessary when isomers may have similar cross-sections. Second, low temperature shortens the mean free path and decreases the neutral buffer gas speeds allowing for more interactions between the ions and the drift gas. Kr2+ study was performed to verify instrument performance. The variable-temperature ion mobility spectrometer was utilized to separate the distonic and conventional ion forms of CH3OH, CH3F, and CH3NH2 and to discriminate between the keto and enol forms of the acetone radical cation. Density functional theory and ab initio calculations were employed to aid in proper identification of separating isomers. Monte Carlo integration tools were also developed to predict ion cross-section and resolution within a buffer gas.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.