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.

Indiana University-Purdue University Indianapolis (IUPUI) / Ion mobility spectrometry is a powerful technique for the study related to molecule. The work of tow major applications are introduced in this paper. The first application is the optimization of parameters in CCS. The accurate calculation of the collision cross section for multiple molecules is a long-time interested topic in the research for substances detection in micro scale. No reliable analytical approach to calculate the collision cross section has been established to date. Different approaches rely on different mechanism will provide different results in significant extent. This work introduce a method for the determination of parameters in the Lennard Jones potential. Experimental data combined with numerical computation was the fundamental strategy during the optimization of the parameters. In the experiment, electrospray is used as the ion source of IMS while a nebulizer was utilized to electrify the aromatic compounds. New parameters show no less accuracy and equal efficiency while can explain the physical meaning of the collision more clearly. The second application is the trace detection of explosives with very low concentration. The detection of explosives is an important topic in security, while the detection will be difficult due to the low vapor pressure of explosives. In this work, two types of devices are designed for the trace detection of explosives at an extremely low concentration. TNT is selected as the explosives in the experiment. The experiment succeed to reach a sensitivity of 1 part per quintillion, and even find out a linear relationship between the logarithm of TNT concentration and TNT vapor pressure.

Ion mobility spectrometry is a powerful technique for the study related to molecule. The work of tow major applications are introduced in this paper. The first application is the optimization of parameters in CCS. The accurate calculation of the collision cross section for multiple molecules is a long-time interested topic in the research for substances detection in micro scale. No reliable analytical approach to calculate the collision cross section has been established to date. Different approaches rely on different mechanism will provide different results in significant extent. This work introduce a method for the determination of parameters in the Lennard Jones potential. Experimental data combined with numerical computation was the fundamental strategy during the optimization of the parameters. In the experiment, electrospray is used as the ion source of IMS while a nebulizer was utilized to electrify the aromatic compounds. New parameters show no less accuracy and equal efficiency while can explain the physical meaning of the collision more clearly. The second application is the trace detection of explosives with very low concentration. The detection of explosives is an important topic in security, while the detection will be difficult due to the low vapor pressure of explosives. In this work, two types of devices are designed for the trace detection of explosives at an extremely low concentration. TNT is selected as the explosives in the experiment. The experiment succeed to reach a sensitivity of 1 part per quintillion, and even find out a linear relationship between the logarithm of TNT concentration and TNT vapor pressure.

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.

Our group has been studying chiral recognition in gas phase using mass spectrometry for more than 10 years. We are interested in gas phase studies of fundamental interactions because the gas phase avoids complications and masking effects that may arise upon solvation. Therefore, the results of gas phase experiments can be directly compared with those of high-level computational studies. In chapter 2, I studied the roles of hydrogen bonding and pi stacking in gas phase chiral recognition between aromatic crown molecules and aromatic amines. High affinity between host and guest doesn't necessarily result in better recognition. If the affinity is too high, both host enantiomers will bind to the chiral guest very tightly so little discrimination is observed. In order to build an efficient chiral recognition system, we need to select a host and guest that have intermediate binding affinity. Hydrogen bonding is another significant factor that controls the host-guest affinity. In the case of host 1, more hydrogen bonds results in better recognition. We also find that the degree of chiral recognition is greater in the gas phase than in solution. Modeling at the B3LYP/6-31G* level is qualitatively correct, but quantitative agreement with experiment is poor. Inspired by Rekharsky's work which shows successful induced chiral recognition with an achiral host (cucurbituril) in solution, we tested the possibility of applying cucurbiturils as gas phase chiral recognition containers in chapter 5. Conferring chirality on cucurbiturils makes the chiral recognition happen in a restricted space, which might strengthen or hinder the discrimination. By comparing our results with Rekharsky's, we showed the role of solvent in this chiral recognition process. In the gas phase, the enantiodiscrimination does not happen between the "leaving MP" and the "approaching" stronger chiral binder. Because hydrophobic effects are absent in the gas phase, it is possible that the hydrophobic methyl substituent of 2-methylpiperazine and the stronger chiral binder might not be simultaneously included inside the cavity. Therefore, we do not observe enantiodiscrimination in gas phase. The dissociation experiment for the CB[7] ternary complex shows that sec-butylamine binds externally to the CB[7] host. Further, the heterochiral diastereomer is more stable than the homochiral diastereomer. This conclusion is consistent with Rekharsky's result in solution. For more than 15 years, the most common ionization method in our lab has been electrospray ionization. However, ESI is subject to problems with ion suppression, especially when the sample is a mixture or it has a high concentration of salt. The easily ionized molecules tend to scavenge the available charges in the spray solution and dominate the resulting ion population even though other compounds may be present in high abundance. Nanoelectrospray usually yields cold ionization, and analyte suppression can be greatly reduced at nanospray flow rates. Therefore, we constructed a porous polymer monolith (PPM) nanospray emitter similar to that described by Oleschuk et al. and characterized the properties of the PPM emitter. This work is described in chapter 3. Our tests show that this PPM nanospray emitter possesses some special analytical properties: decreased ion suppression, quite stable spray, strong signal intensity and good reproducibility in emitter performance. Chapter 4 deals with the application of the new CRAFTI method to negative ions. CRAFTI stands for cross-sectional areas by Fourier transform ICR. The CRAFTI technique measures collision cross sections, providing a probe of the gas phase conformations of supramolecular complexes. Our preliminary work has shown that CRAFTI is applicable to positive ions, so we further demonstrate the application of the newly-developed method to negative ions in this work. Based on the fact that the experimental cross sections correlate linearly with the theoretical values, we have obtained evidence that CRAFTI is a valid method for negative ions. However, some problems remain. First, we are still working to understand the physical meaning of the CRAFTI cross sections. The absolute values we obtain are generally greater than those obtained from momentum transfer cross section calculations modeled in helium. Second, the precision of the measurements (currently about 2-3%) is still larger than we desire. We need to carefully tune the excitation and isolation amplitudes to make the signal strong and monoisotopic for weak ions. CRAFTI is a very promising and attractive method because FT-ICR provides accurate mass-to-charge measurement along with the cross section measurement. In other words, one technique is sufficient to obtain the shape, size and mass of a molecule simultaneously.

<p>    Drift tubes (DT) are prominent tools used in Ion Mobility Spectrometry (IMS) to separate ions in the gas phase due to their difference in mobility. While prominently used for small ions (< 10nm), their use for larger particles (up to 100nm) is limited and can only be attempted at atmospheric pressure due to diffusion. A system that specializes in high sensitivity larger particles (up to 1000nm) is the Differential Mobility Analyzer (DMA), but lacks in resolution (< 10 for particles 30-1000nm). The idea behind this work is to be able to design a new IMS system based on similar<br> principles to the DT but that allows high resolution and sensitivity for a large range of sizes if possible. The primary idea revolves around the principle of non-constant linear fields to try and control the width of the ion packet as it travels through the system. The first attempt was an Inverted Drift Tube (IDT) which lacked sufficient<br> sensitivity. This was followed by the development of the Varying Field Drift Tube (VFDT) which was the first of such systems to perform better than a regular DT, but only marginally. Finally, the last version of the system included a secondary pulse and labeled High Voltage Pulse - Varying Field Drift Tube (HVP-VFDT), which solved<br> some of the issues of the VFDT and was able to achieve resolving powers of 250, 3-5 times higher than regular DT.</p> <p><br>    In the IDT system, a gas flow is used to drive the packet of ions through the drift region while a linearly increasing electric field which is in the opposite direction of the flow is used to slow down the ions and separate them. In this regime it is the largest ions that arrive at the detector first, hence the name Inverted Drift Tube.This technique would allow larger ions and particles to be detected. At the same time, the linear field can be shown to have diffusion constriction (auto-correction) properties, where the broad distributions may be narrowed in the axial direction. However, the gas flow is difficult to control well and the parabolic velocity profile of the gas flow in the tube is a unfavorable factor for the system. </p> <p><br></p> <p>    To avoid the issue of the parabolic velocity but still take into account the VFDT takes the advantage of the diffusion auto-correction, the gas flow is suppressed and a linearly decreasing field is used to drive the ions. By solving the Nernst-Planck equation, we show that the VFDT has a spatial resolving power that is much higher than that of the regular DT. A DT was built and tested using a mixture of tetraalkylammonium salts. The transformation from the raw variable arrival time distribution to collision cross section or mobility diameter is derived and the linear relationship<br> makes it simple for calibration and transformation. A resolving power of over 90 is achieved experimentally although higher resolving powers were expected theory. <br>  <br>  It turns out that the difference between theory and experiments had to do with the fact that in the VFDT, the spatial and time resolving powers are different.This<br> is due to the low drift velocity at the end of the drift tube. To increase this velocity, a high voltage pulse is applied at a certain time depending on the ion/s of interest with a new system, HVP-VFDT. The system was tested numerically and experimentally where several parameters where tested resulting in a higher resolving powers when compared with DT and VFDT systems.The simulation results showed that the transmission efficiency and resolving power can be controlled by raising or lowering the field. Overall, the experimental setup tested reached resolving powers of 250 with moderate gate pulses. The HVP-VFDT system also shows that the distribution may be narrowed over the initial one, something impossible with a real drift tube and<br> opens a myriad of possibilities, including resolving powers of several thousands under low pressure and RF fields. <br>    <br>  The next step will be to couple the system to a Mass Spectrometer which is expected to be completed in the near future. To understand how a DT works with   RF fields and low pressure, a collaboration was done with David Clemmer’s lab and his 4 meter drift tube that can achieve resolutions of 150 in Helium at 4torr. Here, we tested a set of polymers and compared the results to those acquired in Nitrogen with a DMA. The shape and structure of the polymers in the gas phase was studied showing<br> self-similar assemblies that corresponds to a globule with an appendix sticking out. <br>  <br>    </p>

Wide bandgap (WBG) devices and power electronic converters (PEC) that enable the dynamic control of energy and high-power density designs inevitably contain defects including sharp edges, triple points, and cavities, which result in local electric field enhancements. The intensified local electric stresses cause either immediate dielectric breakdown or partial discharge (PD) that erodes electrical insulators and accelerates device aging. With the goal of addressing these dielectric challenges emerging in power-dense applications, this dissertation focuses on 1) modeling the dielectric characteristics of supercritical fluids (SCFs), which is a new dielectric medium with high dielectric strength and high cooling capability; and 2) establishing the optimal fabrication conditions of electrets, which is a new dielectric solution that neutralizes locally enhanced electric fields. In this dissertation, the dielectric breakdown characteristics of SCFs are modeled as a function of pressure based on the electron scattering cross section data of clusters that vary in size as a function of temperature and pressure around the critical point. The modeled breakdown electric field is compared with the experimental breakdown measurements of supercritical fluids, which show close agreement. In addition, electrets are fabricated based on the triode-corona charging method and their PD mitigation performance is evaluated through a series of PD experiments. Electrets are fabricated under various charging conditions, including charging voltage, duration, polarity, and temperature with the goal of identifying the optimal condition that leads to effective PD mitigation. The PD mitigation performance of electrets fabricated based on these charging conditions is further assessed by investigating the impact of various power electronics voltage characteristics, including dv/dt, polarity, switching frequency, and duty cycle. Electret based electric field neutralization approach is further utilized in increasing the critical flashover voltage associated with the surface flashover voltage. Moreover, due to the high mechanical strength of epoxy composites at cryogenic temperatures, in this dissertation, epoxy-based electrets are fabricated as a solution to PD in high temperature superconducting cables. The experimental demonstrations conducted with electret in this dissertation is dedicated for the establishing the electret based electric field neutralization approach as a dielectric solution for the dielectric challenges in power electronics driven systems.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23135号 / 薬科博第134号 / 新制||薬科||15(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 石濱 泰, 教授 松﨑 勝巳, 教授 加藤 博章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Ion mobility spectrometry (IMS) separates ions on the basis of ion-neutral collision cross-sections (CCS, [omega]), which are determined by the geometry or conformation of the ions. The size-based IM separation can be extended to distinguish conformers that have different shapes in cases where shape differences influence the accessible surface area of the molecule. In recent years, IM has rapidly evolved as a structural characterization technique, which has applied on various structural biology problems. In this work, IMS is combined with molecular dynamics simulation (MDS), specially the integrated tempering sampling molecular dynamics simulation (ITS-MDS) to explore the gas-phase conformation space of two molecular systems (i) protonated tryptophan zipper 1 (trpzip1) ions and its six derivatives (ii) alkali metal ion (Na, K and Cs) adducts of gramicidin A (GA). The structural distributions obtained from ITS-MDS are compared well with results obtained from matrix-assisted laser desorption ionization-ion mobility-mass spectrometry (MALDI-IM-MS) for trpzip 1 series and electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) for alkali metal ion adducts of GA. Furthermore, the solvent dependence on conformational preferences of the GA dimer is investigated using a combination of mass spectrometry techniques, viz. ESI-IM-MS and hydrogen/deuterium exchange (HDX)-MS, and MDS. The IM experiments reveal three distinct gramicidin A species, detected as the sodium ion adduct ions, [2GA + 2Na]²⁺, and the equilibrium abundances of the dimer ions varies with solvent polarity. The solution phase conformations are assigned as the parallel and anti-parallel [beta]-helix dimer, and the anti-parallel dimer is the preferred conformation in non-polar organic solvent. The calculated CCS profiles by ITS-MDS agree very well with the experimentally measured CCS profiles, which underscore the utility of the method for determining candidate structures as well as the relative abundances of the candidate structures. The benefit of combining ion mobility measurements with solution-phase H/D exchange is allowing identifications and detail analysis of the solution-phase subgroup conformations, which cannot be uncovered by one method alone.

Proteomic studies aim to identify, quantify and characterise the full complement of proteins in a cell or organism under a defined set of conditions, and are important to our understanding of cellular mechanisms. However, such studies represent a major analytical challenge. A typical proteome analysis involves enzyme-mediated digestion of complex protein mixtures to yield an even more complex mixture of peptides. Combined reverse-phase liquid chromatography and tandem mass spectrometry is then traditionally utilised to ascertain sequence information from the characteristic peptide sequences. Analytical data derived for the peptides are employed as search terms in database searching of protein sequences derived from gene sequences. The extreme complexity of the peptide mixtures analysed means that additional novel approaches are required to fully interrogate the vast number of tandem mass spectra generated, assigning peptide identity and thereby helping to address demanding biological questions. The research reported here aims to further our understanding of both gas phase peptide/peptide fragment ion structure and peptide fragmentation behaviour using a combination of tandem mass spectrometry and ion mobility measurement.To facilitate the determination of peptide ion collision cross section, a novel standard, QCAL-IM, produced using the QconCAT strategy, has been developed to enable calibration of drift time in Travelling Wave Ion Mobility instruments. The standard facilitates empirical determination of the rotationally averaged collision cross section of any peptide/peptide fragment ion that lies within the calibration range encompassed. QCAL-IM was subsequently utilised to determine the collision cross section of a range of peptide ions produced by Lys-C and Lys-N proteolysis of ‘standard’ proteins. Data produced allowed the effect upon gas phase ion conformation through changing the location of the basic residue lysine within a peptide sequence to be assessed.The fragmentation behaviour of peptide ions produced by a variety of digestion regimes during both collision-induced dissociation (CID) and electron transfer dissociation (ETD) has also been extensively studied. The proteases trypsin and Lys-C are those typically utilised during proteomic studies and peptides produced by each have either the basic residues arginine or lysine at their carboxy-terminus. Secondary enzymatic treatment with the exoprotease carboxypeptidase B cleaves these basic residues from the C-terminus. Tandem mass spectrometric analysis of both tryptic/Lys-C peptides and their CBPB truncated analogue highlights that the dominant fragment ion series observed during both CID and ETD is determined, at least in part, by the location of such basic residues.Finally, studies were undertaken to investigate the factors which may promote/inhibit scrambling of peptide fragment ion sequence, which has recently been shown to take place during CID. The effect of modifying the gas phase basicity of the N-terminal amino acid residue is studied through a combination of derivatisation and synthesis of alternative peptide sequences. Increasing the gas phase basicity is shown to inhibit the observed sequence scrambling while promoting concomitant rearrangement/retention of a carboxyl oxygen at the C-terminus to give enhanced formation of bn+H2O product ion species.