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Native mass spectrometry protein structural characterization via surface induced dissociation: instrumentation and applicationsYan, Jing 12 December 2017 (has links)
No description available.
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Small heat shock protein interactions with in vivo partnersCollier, Miranda January 2018 (has links)
Small heat-shock proteins (sHsps) are part of a broad cellular sys- tem that functions to maintain a stable proteome under stress. They also perform a variety of regulatory roles at physiological conditions. Despite the multitude of sHsp targets, their interactions with partners are not well understood due to highly dynamical structures. In this thesis, I apply a variety of biophysical and structural approaches to examine distinct interactions made by the abundant human sHsps αβ-crystallin and Hsp27. First, I find that αβ-crystallin binds a cardiac-specific domain of the muscle sarcomere protein titin. A cardiomyopathy-causative variant of αβ-crystallin is shown to disrupt this interaction, with demonstrated implications for tissue biomechanics. Next, I investigate the conformation and unfolding behaviour of another sarcomere-associated protein, filamin C, finding support for the hypothesis that it is mechanosensitive. This leads into an interrogation of the interaction between filamin C and Hsp27, which we find is modulated by phosphorylation of Hsp27. This modulation only manifests during filamin C unfolding, pointing toward a protective chaperoning mode against over-extension during mechanical stress. This finding is bolstered by up-regulation and interaction of both proteins in a mouse model of heart failure. I establish a system for similar studies of a third sHsp, cvHsp, which is muscle-specific and implicated in various myopathies but scantly understood at the molecular level compared to αβ-crystallin and Hsp27. Finally, I probe the stoichiometries and kinetics of complexes formed between αβ-crystallin and Hsp27 themselves, which co-assemble into a highly polydisperse ensemble. This involved the development of a high-resolution native mass spectrometry method for disentangling heterogeneous systems. Together these findings add to our understanding of the roles and mechanisms of ATP-independent molecular chaperones.
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Characterization of Peptides, Proteins, and Protein Complexes using Infrared Multiphoton Dissociation Spectroscopy, Ion Mobility Spectrometry, and Surface-induced Dissociation Mass SpectrometryPanczyk, Erin Michelle 01 October 2021 (has links)
No description available.
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DEVELOPMENT OF GAS-PHASE ION/ION REACTIONS FOR CHARACTERIZING PROTEIN AND PEPTIDE IONSAnthony Marcel Pitts-Mccoy (11024205) 23 July 2021 (has links)
<p>Mass spectrometry-based gas-phase ion/ion reactions have
grown considerably in the last decade. Their applications range from structural
elucidation, instrument calibration, and spectral deconvolution. One field that
has been amenable to these methods is proteomic studies. Proteins and peptides
have grown as candidates for biomarkers and vaccines. Proteins are vastly
different with mass ranging from 1 kDa to well over 1 MDa and various types of
post translational modifications. The structural heterogeneity that proteins
can exhibit demonstrates the need for high resolution mass spectrometry
methods. The combination of native mass
spectrometry and soft ionization sources allow for preservation of structures
seen in solution as analytes enter the gas phase. By developing methods that
probe these structures, the information gathered can be related to the native
structures in solution. Here I show, gas
phase ion/ion reactions that can be utilized for location of salt bridge
structures, gas-phase crosslinking of homo and heterodimer protein complexes,
and mass determination of large (>800 kDa) protein complexes. These methods
allow for greater control, faster data acquisition, and minimal sample
preparation. These methods were developed on modified Sciex TripleTOF 5600 and
4000 QTRAP tandem mass spectrometers.</p>
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Native Mass Spectrometry for Characterization of Protein:Protein and Protein:RNA ComplexesJia, Mengxuan January 2019 (has links)
No description available.
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Use of fluorescence resonance energy transfer (FRET) to elucidate structure-function relationships in archaeal RNase P, a multi-subunit catalytic ribonucleoproteinMarathe, Ila Abhijit January 2021 (has links)
No description available.
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Characterization of Proteins and Protein Complexes by Online Chromatographic Separations and Direct Infusion Native Mass SpectrometrySahasrabuddhe, Aniruddha 02 August 2018 (has links)
No description available.
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Investigation of the Editing Mechanism of Prolyl-tRNA Synthetase and Characterization of RNase P Variants by Mass SpectrometryTanimoto, Akiko January 2016 (has links)
No description available.
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Dynamic combinatorial mass spectrometry for 2-oxoglutarate oxygenase inhibitionDemetriades, Marina January 2013 (has links)
In the last decade, dynamic combinatorial mass spectrometry (DCMS) with protein targets has emerged as a promising method for the identification of enzyme-inhibitors. 2-Oxoglutarate (2OG) oxygenases are involved in important biological processes related to many diseases; several human 2OG oxygenases are targeted for pharmaceutical intervention. This thesis describes inhibition studies on three 2OG oxygenases using DCMS and structure activity relation (SAR) studies. Disulphide based DCMS was used for the identification of N-oxalyl based lead inhibitors for the 2OG oxygenase AlkB from Escherichia coli. Crystallographic analyses of AlkB with a lead inhibitor assisted in the design of a second generation of inhibitors using N-oxalyl, pyridyl and quinolinyl scaffolds. Crystallographic and kinetic data of three potent and selective AlkB inhibitors validates the DCMS approach for the development of 2OG oxygenase inhibitors. The hypoxia inducible factor hydroxylase, prolyl hydroxylase domain 2 (PHD2), was then used as the model enzyme for the development of a novel DCMS approach employing the reversible reaction of boronic acids with diols to form boronate esters. The ‘boronate’ DCMS method was used to identify pyridyl- substituted lead compounds. Further modification of the pyridine scaffold, based on structural analyses, led to the development of highly potent and selective PHD2 inhibitors. To identify inhibitors for the fat mass and obesity associated protein (FTO), another 2OG oxygenase, an inhibition assay was developed. The inhibition assay was used in conjunction with a differential scanning fluorimetry (DSF) binding assay to identify isoquinolinyl and pyridyl inhibitor scaffolds, related to those used in the DCMS studies. FTO complexed structures of these compounds, and with a natural product anthraquinone, enabled the design and synthesis of new inhibitors that are both co-substrate and substrate competitors of FTO. One such compound proved to be a potent FTO inhibitor with improved selectivity over other 2OG oxygenases. Overall, the work validates the use of DCMS methods for the development of potent and selective inhibitors for 2OG oxygenases, and by implication of other enzyme families.
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Analyse biochimique et par spectrométrie de masse d'un complexe ribonucléoprotéique d'export du VIH-1 / Biochemical and mass spectrometry analysis of an HIV-1 ribonucleoprotein export complexOliva, Mizar Francesca 23 May 2017 (has links)
Une étape importante du cycle viral du virus de l'immunodéficience humaine (VIH) est l'export nucléaire de transcrits viraux incomplètement épissés, incluant le génome viral ARN. Ce processus fait intervenir la protéine virale de liaison à l'ARN Rev. Dans le noyau, Rev interagit avec les transcrits viraux non épissés et partiellement épissés en s'oligomérisant sur une séquence intronique de 350 nucléotides, appelée Element de Response à Rev (RRE). Rev recrute également le facteur d'export cellulaire CRM1 et la petite GTPase Ran pour former le complexe d'export RRE/Rev/CRM1/Ran. Connaître l'architecture 3D de ce complexe ribonucléoprotéique fournirait des informations utiles pour une meilleure compréhension de l'export des ARN du VIH incomplètement épissés. Cependant, les détails moléculaires de ce complexe sont mal connus ; en particulier, la stœchiométrie des molécules Rev et CRM1 liées au RRE est en discussion.Mon doctorat vise à étudier l'architecture du complexe RRE/Rev/CRM1/Ran. Dans le cadre de ce travail, j'ai utilisé des essais biochimiques et cellulaires pour caractériser les interactions entre CRM1 et Rev et entre Rev et RRE. La majorité de mes efforts ont porté sur l'étude de ces interactions par spectrométrie de masse (MS) en condition native, une méthode puissante pour déterminer la stœchiométrie de complexes macromoléculaires. J'ai mis en place des protocoles pour la préparation à grande échelle d'un fragment du RRE de 66 nucléotides (IIABC), portant un site de liaison Rev de haute affinité, protocoles que j'ai ensuite adaptés à l'analyse de IIABC par MS en condition native. Comme Rev a tendance à s'agréger et à précipiter en solution, j'ai également conçu une forme mutante de Rev (Rev*) permettant de contourner ces problèmes. L'analyse des complexes IIABC/Rev* par électrophorèse sur gel natif confirme l'oligomérisation de Rev* sur l'ARN. Après d'intenses optimisations, j'ai obtenu des spectres MS en condition native de haute qualité, révélant que IIABC lie jusqu'à 6 monomères Rev*. De plus, j'ai reconstitué un complexe à 4 partenaires IIABC/Rev*/CRM1/Ran et j'ai réussi à déterminer sa masse et sa stœchiométrie par MS en condition native, une tâche techniquement difficile. Des efforts supplémentaires pour analyser le RRE seul et en complexe avec Rev de type sauvage ont également généré des spectres informatifs, alors que l'analyse du complexe intact RRE/Rev/CRM1/Ran a été plus compliquée. Ces résultats illustrent les forces et les limites de la spectrométrie de masse en condition native et son potentiel pour son développement futur en tant qu'outil d'analyse des complexes de ribonucléoprotéines. / An important step in the life cycle of human immunodeficiency virus (HIV) is the nuclear export of incompletely spliced viral transcripts, including the replicated viral RNA genome. This process is mediated by the viral RNA-binding protein Rev. In the nucleus, Rev recognizes unspliced and partially spliced viral transcripts by multimerizing on a 350-nucleotide intron sequence, the Rev-response element (RRE). Rev then recruits the host cell export factor CRM1 and the small GTPase Ran to form the RRE/Rev/CRM1/Ran export complex. Knowledge of the 3D architecture of this ribonucleoprotein complex would provide important insights into how unspliced viral RNA export is achieved. However, the molecular details of this complex are poorly understood. In particular, the stoichiometry of Rev and CRM1 molecules bound to the RRE is under debate.My Ph.D. project aims to investigate the architecture of the RRE/Rev/CRM1/Ran complex. As part of this work, I used biochemical and cell-based assays to characterize the interactions between CRM1 and Rev and between Rev and the RRE. The majority of my efforts focused on investigating these interactions by native mass spectrometry (MS), a powerful method for determining the stoichiometry of macromolecular complexes. I set up protocols for the large-scale preparation of a 66-nucleotide RRE fragment (IIABC) bearing a high-affinity Rev binding site, and adapted these for compatibility with native MS analysis. Because Rev tends to aggregate and precipitate in solution, I engineered a mutant form of Rev (Rev*) to overcome this problem. Analysis of IIABC/Rev* complexes by native gel electrophoresis confirms multimerization of Rev on the RNA. After extensive optimization, I obtained high-quality native MS spectra of these complexes, revealing that IIABC binds up to 6 Rev* monomers. Furthermore, I reconstituted a 4-species complex, IIABC/Rev*/CRM1/Ran, and succeeded in determining its mass and stoichiometry by native MS – a technically challenging task. Additional efforts at analyzing the intact RRE and complexes with wild-type Rev have also yielded informative spectra, while analysis of the intact RRE/Rev/CRM1/Ran holo-complex has had more limited success. These results illustrate the strengths and limitations of native mass spectrometry and its potential for future development as a tool for analyzing ribonucleoprotein complexes.
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