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This dissertation presents extensive structural studies of gas-phase peptide fragment ions, with a specific focus on b₂⁺ ions. Fragment ion structures can provide important insights into peptide fragmentation mechanisms. Based on the structures formed, information about the preference of competing b ion formation pathways can be obtained. b₂⁺ ion structures are of interest because of their large relative abundances in MS/MS spectra, which are difficult to predict. Prior to this work, only a few b₂⁺ ion structures were determined; these systems featured only aliphatic residues and all formed oxazolones. The work presented herein examines the influence of basic, acidic, and backbone-attached sidechains on peptide fragmentation mechanisms, as revealed by the resulting b₂⁺ fragment ion structure(s) formed. Specifically, the structures of several histidine, aspartic acid, and proline-containing b₂⁺ ions are determined by using action IRMPD spectroscopy, fragment ion HDX, and DFT calculations. The structures of a series of histidine analogue-containing b₂⁺ ions reveal that the location and availability of the pi-nitrogen is essential for diketopiperazine formation. The histidine sidechain bulk or strain interferes with the complete trans-cis isomerization required for diketopiperazine formation, so the oxazolone structure is also present. Xxx- Pro b₂⁺ ions favor oxazolone formation with aliphatic N-terminal residues. HP favors the diketopiperazine, combining the histidine effect and the proline cis conformation propensity. For Xxx-Asp b₂⁺ ions, aspartic acid significantly influences b₂⁺ ion structure only with an N-terminal histidine or lysine; both HD and KD form a mixture of oxazolone, anhydride, and diketopiperazine structures, presenting the first spectroscopic evidence for the anhydride b₂⁺ion structure. The HA and AH b₂⁺ ions feature the same structures, but HP and PH do not, showing that residue position matters. Additionally, while relative intensities and HDX rates featured some fluctuation, peptide precursor composition differences did not alter the mixture of b₂⁺ ion structures formed for a given b₂⁺ ion. To complement existing gas-phase structural methods, the utility of a new technique, QCID-HDX-IRMPD, was applied to m/z 552.28 from YAGFL-OH. Both the standard b₅⁺ fragment ion and an isobaric non-C-terminal water loss ion are present. Without separation of these isomers, MS/MS spectral interpretation would be complicated.

Mass spectrometry has frequently been employed in the analysis of biologically relevant molecules; however, mass spectrometry alone may not always be sufficient for the differentiation and characterization of isomeric and isobaric ions. In this work, infrared multiple photon dissociation (IRMPD) spectroscopy and ion mobility spectrometry (IMS) were evaluated as complementary techniques for the characterization and separation of isomeric and isobaric ions of biological relevance. In the first project, analysis of experimental IRMPD spectroscopy data shows that this technique is useful in the differentiation of hydroxyproline isomers. Absorption bands allow for the differentiation of three isomeric species: 1640 cm-1 (trans-4-hydroxyproline), 1718 cm-1 (cis-4-hydroxyproline), and 1734 cm-1 (cis-3-hydroxyproline). In the second project, theoretical CCS and IR spectroscopy predictions of isobaric modified amino acids and isomeric drugs have been carried out as predictions of IMS and IRMPD spectroscopy suitability. Preliminary IMS measurements suggest that the CCS predictions are at least qualitatively useful.

Abstract Experimental synchrotron radiation induced electron- and ion spectroscopies together with electron-ion and ion-ion coincidence techniques as well as electron energy loss spectroscopy have been used to study the electronic properties of several vapor phase samples. In this thesis studies of the electronic structure and fragmentation of Sb4 clusters, photo- and Auger electron spectroscopy of atomic Si and Pb as well as ultra high resolution VUV absorption of vapor phase KF molecules have been performed. The instrumentation and techniques used in the studies, especially the electron energy loss apparatus and the newly built ultra high resolution FINEST beamline branch, are presented.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22659号 / 工博第4743号 / 新制||工||1741(附属図書館) / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 山田 啓文, 教授 川上 養一, 准教授 杉山 和彦 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

<p>The work presented here employs cryogenic ion spectroscopy for the study of protein structure, kinetics, and dynamics. The main technique used is IR-UV double resonance spectroscopy. Here peptide ions are generated through nano electrospray ionization, guided into a mass spectrometer, mass selected, and then guided into a cryogenically held octupole ion trap. Ions are subsequently cooled to their vibrational ground state through collisions with 5 K helium allowing for high resolution IR and UV spectra to be recorded. The IR spectra are highly sensitive to an ion’s conformation, and the well resolved UV spectra provides a means generate conformer specific IR spectra. With the use quantum mechanical calculations, it is possible to calculate the vibrational spectra of candidate structures for comparison with experimental spectra. Strong correlations between theory and experiment allow for unambiguous structural assignments to be made.</p> <p> Structural studies are performed on β-turn motifs and well as salt-bridge geometries. Beta-turns are a commonly occurring secondary structure in peptides and proteins. It is possible to artificially encourage the formation of this secondary structural element through the incorporation of the D-proline (^DP) stereoisomer followed by a gly or ala residue. Interestingly, the L-proline (^LP) stereoisomer is seen to discourage the formation of beta turn structure. Here were probe the inherent conformational preferences of the diastereomeric peptide sequences YA^LPAA and YA^DPAA. The findings agree with solution phase studies, the ^DP sequence is observed to adopt a beta turn however, the ^LP sequence is found to undergo a sterically driven <i>trans</i> à <i>cis</i> isomerization about the proline amide bond. We find the energetics associated with this unfavorable interaction and show the ability to reverse it by proper substitution of Ala₂ for a Gly.</p> <p>The studies directed towards gas phase salt bridges have been limited to single amino acids or dipeptides. Generally, these species are ionized using a metal ion or adducted with water or excess electrons in order to stabilize a zwitterionic motif. Here we take the first look at a salt bridge motif incorporated into polypeptide in order to understand how the solvation from the secondary structure can aid in stabilizing these motifs in non-polar environments. We find a unique salt bridge motif in the YGRAR sequence in which the tyrosine OH acts as a neutral bridge to form a network between the C-terminal arginine and the ion pair formed between the central arginine and C-terminal carboxylate group. This binding motif has not been discussed in literature and appears as an important structural element in non-polar environments as all salt bridge character is lost upon substituting Tyr for Phe. We are the process of mining the PDB for these types of interactions. </p> <p>To better understand how cryo-cooling impacts the resulting population distribution at 10 K we measured the distribution among the two major conformation of the YGPAA ion. This was carried out using population transfer spectroscopy. In this method conformational isomerization is induced vis single conformer infrared excitation. The change in population can be related to the final population distribution at 10 K. With this number, we were able to develop a cooling model to simulate the change in the distribution as a function of cooling. The cooling rates, were experimental established, and the isomerization rates and starting population were theoretically derived through RRKM and thermodynamic calculations. With these parameters and cooling model, we found that the room temperature population distribution is largely preserved. When isomerization events involve breaking a hydrogen bond, they become too slow to complete with the cooling time scale of the experiment, effectively freezing in the room temperature structures. These are important physical parameters to characterize when performing structural studies at 10 K.</p> <p>Finally, we demonstrate a 2-Color IRMPD technique that is able to generate linear spectra at varied temperatures. This is in sharp contrast to traditional IRMPD which results in non-linear and skewed spectra. The importance of generating linear spectra when making structural assignments is highlight by comparing the performance between both techniques. Furthermore, with this technique we show the ability to record the spectra of ion prepared with high internal energies. This provides spectroscopic snapshots of the unfolding events leading to dissociation. Overall, the versatility of this technique to record ground state spectra comparable to IR-UV DR, to record linear spectra at room temperature, and to probe dynamics proves this technique to be useful in the field of ion spectroscopy.</p>

Este trabalho conta com um apanhado geral da técnica de espectrometria de massas acoplada a espectroscopia vibracional de íons, suas aplicações, limitações, desenvolvimento e embasamento teórico. Para tal é apresentado um resumo da teoria quântica aplicada à espectroscopia vibracional, natureza da luz, eletromagnetismo e ótica de sistemas de OPO/OPA, laser de gás e de estado sólido. A seção experimental é dividida em duas. A primeira apresenta o acoplamento físico do espectrômetro de massas às fontes de radiação laser, juntamente com os softwares para permitir comunicação entre esses módulos. Na segunda parte, são apresentados espectros IRMPD de amostras padrão para comparação e validação com a literatura bem como espectros inéditos para exemplificar o uso da técnica de espectroscopia de íons em sistemas de interesse biológico, como as bases nitrogenadas do DNA. / This master thesis presents a general overview of the mass spectrometry technique coupled to vibrational ion spectroscopy, its applications, limitations, development and theoretical basis. For that, a summary of the quantum theory applied to vibrational spectroscopy, the nature of light, electromagnetism and optics of OPO/OPA, gas discharge and solid-state laser systems is shown. The experimental section is divided in two, the first presents the physical coupling of the mass spectrometer to the laser sources, along with softwares which allows the communication between these modules. In the second part, some IRMPD spectra of standard samples are presented for comparison and validation with the literature as well as unpublished spectra to exemplify the use of the ion spectroscopy technique in systems of biological interest, such as the DNA bases.

Electronic excitation triggered by the absorption of light enables numerous chemical, physical and biological processes and transformations. Accordingly, full control over the processes involving excited molecules requires an in-depth knowledge of electronic UV/vis spectra and potential energy surfaces. Unsurprisingly, most electronic spectra are acquired in the condensed phase in which molecules are dissolved and most transformations occur. However, our knowledge of excitation, transformations and processes at the level of isolated molecules is still limited, partly because such studies require unconventional experimental approaches and equipment. This Thesis describes experimental methods for recording electronic spectra of isolated molecules in the gas phase by ion spectroscopy, which combines mass spectrometry with optical spectroscopy. Using these methods, experimental factors which affect the electronic excitation and therefore the electronic spectra of ions were determined and evaluated for various fluorescent xanthene dyes, iron-containing complexes and molecular pho- toswitches. Furthermore, factors which govern photochemical processes, such as photo- oxidation, photoreduction and photoisomerization, were also analyzed in detail, with surprisingly different outcomes from previous studies...

Traditional solution and solid state approaches (Nuclear Magnetic Resonance, X-Ray Crystallography) are methods of choice when analysing both biological and inorganic analytes. However, the characterisation of transient species, often encountered in self-assembling systems, is difficult. Such systems rarely produce crystals of high quality and due to their dynamic nature; their structures are difficult to study with NMR. Hyphenated gas phase methods which rely on mass spectrometry detection offer simultaneous structural analysis and direct stoichiometry measurement. As a consequence, it is possible to investigate specific, non-interacting molecules and molecular complexes in an isolated environment. This thesis focuses on the development and applications of two such methods - ion mobility mass spectrometry (IM-MS) and cold ion spectroscopy. IM-MS measurements yield a so called collisional cross sectional area (CCS). This parameter can be pictured as a rotationally averaged, shadow projection of a molecule structure. When correlated with the ion abundance, a CCS distribution yields intuitively interpretable information about the conformational preferences of an isolated molecule. Although indispensable in describing a "global" geometrical structure, the CCS parameter itself provides a limited insight into the local structural features of the assembly. Ion spectroscopy, both in the UV and IR regions, can provide an extra layer of highly descriptive information. Here, we present several cases where the above techniques have been applied. With the aid of IM-MS, we have analysed the geometry of inorganic supramolecular assemblies, highlighting the stability of particular metal-ligand interactions. Using cold ion spectroscopy, we have assessed the fine structural information of self-assembled oligomers of an amyloidogenic peptide. We correlated spectral features of isolated oligomers to features observed in the mature fibrils; therefore attempting to delineate the events in early stages of amyloidogenic aggregation. A major part of this report focusses on technological aspects of the design and development of a high resolution, variable temperature ion mobility mass spectrometer (VT-IM-MS). The thermal stability of molecules is a vital aspect in industrial process development and formulation science. Solution phase Differential Scanning Calorimetry (DSC) is a widely applied technique, allowing to monitor reversibility of thermally induced conformational transitions, a key aspect in protein folding analysis. The instrument reported here aims to provide parallel information about gaseous ions, with a particular focus on protein ions. Capabilities of the newly built instrument have been tested using small, rigid molecules, a small protein and a large multiprotein complex.

Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2017-11-21T12:03:53Z No. of bitstreams: 0 / Made available in DSpace on 2017-11-21T12:03:53Z (GMT). No. of bitstreams: 0 / O estudo objetivou desenvolver uma novo método de extração para analisar os poluentes orgânicos persistentes (POPs), no sedimento da Represa Billings em São Paulo, via cromatografia a gás com espectrometria de massas (GC/MS). O Tratado de Estocolmo realizado na Suécia em 1972, patrocinado pela ONU, possuindo 113 países participantes, inclundo o Brasil objetivou a eliminação de menos doze POPs, colaborando com a questão ambiental. Os POPs são compostos que persistem no meio ambiente e são altamente estáveis, podendo ultrapassar a barreira placentária. Neste trabalho foram abordados o Aldrin, Endrin, Dieldrin, DDT. DDE, DDD e Heptacloro. Para garantir a confiabilidade dos resultados analíticos, foram realizados ensaios de validação do método, baseados nas diretrizes do INMETRO. O método de extração dos analitos foi o Quechers, obtendo resultados de recuperação entre 40 e 120% para todos os POPs analisados neste trabalho, valores aceitáveis para matrizes complexas como o sedimento. Os limites de detecção e quantificação fora, 0,3 e 0,5 &mu;g/kg respectivamente. As amostras analisadas não apresentaram contaminação significativa por POPs perante a Legislação Brasileira vigente. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP