The amino acid sequence of a protein determines its 3D fold, the ease with which its native structure is formed, its function, the conformational preferences sampled and the tendency to interact with itself (aggregation) and binding partners. In addition, certain conformational preferences can lead to dysfunction resulting in different diseased states in organisms. All of these conformations can be described by a protein's energy landscape; a native (functional) state being localised at the energy minimum. As protein dynamics is crucial to function it is important to monitor the sampling of different conformations. Thus the work in this thesis reports on two methods for monitoring protein conformation and conformational change in the gas phase using ion mobility mass spectrometry (IM-MS). The measurement from IM-MS methods allow the determination of a collision cross section (CCS) which is an indicator of a molecule's 3D shape. First, the effect of charge on protein structure is investigated by manipulation of protein charge, post electrospray ionisation (ESI), by exposure to radical anions of the electron transfer reagent, 1,3-dicyanaobenzene; the charge reduced products formed are the result of electron transfer to the charged protein without any dissociation (ETnoD). IM-MS is used to monitor the conformational preferences of the altered and unaltered precursor and its products. Secondly, intermediate (transient) conformers are formed by activating the charged protein in the source region of an instrument post ESI. Activation of the protein precursor allows the sampling of different conformational preferences after energetic barriers have been overcome; IM-MS following activation allows for the monitoring of protein conformational change before and after. Further, variable temperature (VT) IM-MS allows for the deduction of intermediate structures with a focus on measurements at cryogenic temperatures whereby intermediate structures can be 'frozen out' post activation; intermediate structures which would otherwise anneal out at room temperature. With both methods a range of conformer populations are mapped for different protein molecules sampled upon different energetic inputs (via activation) and the disruption of intramolecular neutralising contacts/salt bridges (via charge reduction) one of the main interactions responsible for maintaining the structural integrity (3D fold) of proteins.

IM-MS is a 2-D technique which provides separations based on ion shape (ion-neutral collision cross-section, Ω) and mass (m/z ratio). Ion structures can be deduced from the measured collision cross-section (Ωmeas) by calculating the collision cross-sections (Ωcalc) of candidates generated by molecular dynamics (MD) and compared with the experiment results. A database of Ωs for singly-charged peptide ions is presented. Standard proteins are digested using different enzymes (trypsin, chymotrypsin and pepsin), resulting in peptides that differ in amino acid composition. The majority (63%) of the peptide ion correlates well with the globular structures, but some exhibit Ωs that are significantly larger or smaller than the average correlation. Of the peptide ions having larger Ωs, approximately 71% are derived from trypsin digestion and most of the peptide ions that have smaller Ωs are derived from pepsin digestion (90%). We use computational simulations and clustering methods to assign backbone conformations for singly-protonated ions of the model peptide (NH2-Met-Ile-Phe-Ala-Gly-Ile-Lys-COOH) formed by both MALDI and ESI and compare the structures of MIFAGIK derivatives to test the ‘sensitivity’ of the cluster analysis method. Cluster analysis suggests that [MIFAGIK + H]+ ions formed by MALDI have a predominantly turn structure even though the low energy ions prefer partial helical conformers. Although the ions formed by ESI have Ωs that are different from those formed by MALDI, the results of cluster analysis indicate that the ions backbone structures are similar. Chemical modifications (N-acetyl, methylester, as well as addition of Boc or Fmoc groups) of MIFAGIK alter the distribution of various conformers, the most dramatic changes are observed for the [M + Na]+ ion, which show a strong preference for random coil conformers owing to the strong solvation by the backbone amide groups. Ωmeas of oligodeoxynucleotides in different length have been measured in both positive and negative modes. For a given molecular weight and charge state, Ωmeas of the oligodeoxynucleotide ions are smaller than those of the peptides, indicating their different packing efficiency. A novel generalized non-Boltzman sampling MD has been utilized to investigate the gas-phase ion conformations of dGGATC based on the free energy values. Theory predicts only one low-energy conformer for the zwitterionic form of dGGATC- while dGGATC+ ions have several stable conformers in both canonical and zwitterionic form in the gas phase, in good agreement with the experiment.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22505号 / 農博第2409号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5285(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 宮川 恒, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(地球環境学) / 甲第22617号 / 地環博第196号 / 新制||地環||38(附属図書館) / 京都大学大学院地球環境学舎地球環境学専攻 / (主査)教授 藤井 滋穂, 教授 梶井 克純, 准教授 田中 周平 / 学位規則第4条第1項該当 / Doctor of Global Environmental Studies / Kyoto University / DFAM

No description available.

This study is divided into three parts. Part I deals with the mechanism of the self-initiated polymerization (or thermal polymerization) of styrene in the gas phase. In this work, we present the first direct evidence for the thermally self-initiated polymerization of styrene in the gas phase. Our approach is based on on-line analysis of the gas phase Oligomers by mass-selected ion mobility. The mobility measurements provide structural information on the ionized oligomers based on their collision cross-sections (Ω) which depend on the geometric shapes of the ions. Theoretical calculations of possible structural candidates of the Oligomers ions are then used to compute angle averaged Ω for comparison with the measured ones. The agreement between the measured and calculated Ω of the candidate structures provides reliable assignments to the structures of the oligomers. Furthermore, collisional-induced dissociations of the mass-selected oligomer ions provide further support for the structures obtained from the mobility measurements. Our results indicate that the gas phase polymerization of styrene proceeds via essentially the same initiation mechanism (the Mayo mechanism) as in condensed phase polymerization. The structural evidence, the mechanism of formation and the observed fragmentation pathway of the growing dimers and trimers in the gas phase are presentedIn Part II the solvation of a variety of metal cations by benzene clusters have been studied using laser vaporization, cluster beam and time-of-flight mass spectrometry techniques. In this work strong magic numbers were observed for clusters containing 10, 13 and 14 benzene molecules depending on the nature of the metal cation involved. The metal cations exhibiting preference solvation by 14 benzene molecules show a strong tendency to form sandwich structures with two benzene molecules. The interpretation of these results in view of the proposed structures and the growth patterns of the clusters are presented. In Part III, the work is focused on the investigation of the intracluster ion molecule reactions following the generation of Mg+ within the polar clusters (water, methanol, ether and acetonitrile).

No description available.

No description available.

No description available.