Tesis: "Quantitative proteomic analysis"

1

Eberl, Hans Christian. "Quantitative proteomic analysis of chromatin associated protein complexes". Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-153101.

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2

Chiu, Han-Chen. "High-throughput quantitative proteomic analysis of dengue virus infected cells". Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.687071.

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The four serotypes of dengue virus (DENV 1-4) cause the most important arthropod-borne viral disease of humans. Dengue is a global health concern with up to 390 million human infections estimated to occur annually. Despite intensive research, the pathogenesis of dengue is not well understood. The use of high-throughput transcriptomic and small interfering RNA approaches has led to the identification of cellular proteins and pathways that are required for, and modulated by, the DENV lifecycle. In comparison to other high-throughput approaches, high-throughput proteomic analysis has not been applied to the investigation of the DENV -host protein interaction. In this study, for the first time, ~table isotope labelling by ~mino acids in fell culture (SILAC) combined with high throughput mass spectrometry (MS) has been used to examine the host cell response to DENV infection. Initially, SILAC-MS analysis was used to investigate the changes that occurred in the proteome of human alveolar epithelial A549 cells in response to DENV infection. Nuclear and cytoplasmic fractions prepared from mock and DENV -2 infected A549 cells were analysed. 2115 and 3098 proteins in nuclear and cytoplasmic fractions respectively were identified and quantified in both the mock and DENV -2 infected A549 cells. The SILAC-MS results were subjected to bioinformatics analysis and validated for eight selected proteins by Western blot and immunofluorescence analysis. Two of the selected proteins, ELKS/Rab6-interactinglCAST family member 1 (ERC 1) and PRA 1 family protein 2 (PRAF2), significantly decreased during infection and were investigated in more detail to determine their relevance to the DENV lifecycle. Knockdown of ERCI but not PRAF2 inhibited DENV -2 replication whereas overexpression of PRAF2 but not ERCI inhibited DENV-2 infection. Co-immunoprecipitation analysis combined with SILAC-MS revealed that ERCI and PRAF2 interacted with the DENV NS5 and NS 1 respectively and cellular proteins involved in trafficking, suggesting that DENV may modulate intracellular transport processes during its release. Comparative analysis of DENV -2 and DENV -4 infected human hepatocyte Huh-7 cells was then done by SILAC-MS. The SILAC-MS results identified common and cell specific pathways that were modulated by DENV infection and identified cellular proteins that were differentially effected by DENV -2 and DENV -4 infection. Furthermore, the analysis of cell specific pathways that were modulated by DENV infection was done by comparison ofDENV-2 infected A549 and DENV-2 infected Huh- 7 cells. Overall the work described in this thesis demonstrated that SILAC-MS is a powerful approach for the analysis of changes in the host proteome upon DENV infection. A number of novel protein changes were identified that can now be furher examined to increase our understanding of DENV replication and identify targets against which antiviral strategies can be developed.

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3

Kaneva, Iliyana. "Quantitative proteomic analysis of kinase and phosphatase substrates in Candida albicans". Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/17558/.

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The ability of the fungus Candida albicans to switch interchangeably between yeast and hyphal forms of growth contributes significantly to its pathogenesis. This morphogenetic shift occurs in response to environmental changes and it is accomplished by a complex network of signal transduction pathways. Protein kinases and phosphatases are important messengers in these pathways and several of them have been directly implicated in controlling C. albicans morphogenesis. Kinases and phosphatases (KP) are enzymes that modulate the function of their substrates via reversible phosphorylation and dephosphorylation, respectively. While the specificity of KP is tightly controlled, some enzymes can target a huge number of proteins and have a master regulatory role over various cell processes. The function of KP in C. albicans is poorly understood and methods for global analysis of KP interactions have not been adapted to this organism. This study developed a protocol for large scale analysis of protein interactions in C. albicans using immunoprecipitation and SILAC in conjunction with quantitative mass spectrometry analysis. The protocol was successfully applied for identification of Cdc14 interactors using the substrate-trapping mutant Cdc14C275S. Cdc14 is a phosphatase required for proper hyphal formation, cytoskeletal organisation and cell separation at the end of mitosis. This study reveals over 100 potential substrates of Cdc14 and new roles of the phosphatase in DNA damage repair, DNA replication, chromosome segregation and transcription regulation. In addition, experiments were performed separately with both yeast and hyphae allowing for direct comparison of Cdc14 interactome between both forms. Many of the identified proteins have unknown function and the significance of these putative interactions remains to be found.

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4

McQueen, Peter. "Alternative strategies for proteomic analysis and relative protein quantitation". Wiley-VCH, 2015. http://hdl.handle.net/1993/30850.

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The main approach to studying the proteome is a technique called data dependent acquisition (DDA). In DDA, peptides are analyzed by mass spectrometry to determine the protein composition of a biological isolate. However, DDA is limited in its ability to analyze the proteome, in that it only selects the most abundant ions for analysis, and different protein identifications can result even if the same sample is analyzed multiple times in succession. Data independent acquisition (DIA) is a newly developed method that should be able to solve these limitations and improve our ability to analyze the proteome. We used an implementation of DIA (SWATH) to perform relative protein quantitation in the model bacterial system, Clostridium stercorarium, using two different carbohydrate sources, and found that it was able to provide precise quantitation of proteins and was overall more consistent in its ability to identify components of the proteome than DDA. Relative quantitation of proteins is an important method that can determine which proteins are important to a biochemical process of interest. How we determine which proteins are differentially regulated between different conditions is an important question in proteomic analysis. We developed a new approach to analyzing differential protein expression using variation between biological replicates to determine which proteins are being differentially regulated between two conditions. This analysis showed that a large proportion of proteins identified by quantitative proteomic analysis can be differentially regulated and that these proteins are in fact related to biological processes. Analyzing changes in protein expression is a useful tool that can pinpoint many key processes in biological systems. However, these techniques fail to take into account that enzyme activity is regulated by other factors than controlling their level of expression. Activity based protein profiling (ABPP) is a method that can determine the activity state of an enzyme in whole cell proteomes. We found that enzyme activity can change in response to a number of different conditions and that these changes do not always correspond with compositional changes. Mass spectrometry techniques were also used to identify serine hydrolases and characterize their expression in this organism.
February 2016

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5

Eberl, Hans Christian [Verfasser] y Matthias [Akademischer Betreuer] Mann. "Quantitative proteomic analysis of chromatin associated protein complexes / Hans Christian Eberl. Betreuer: Matthias Mann". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1031380728/34.

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6

Yousuf, Amjad. "High-throughput quantitative proteomic analysis of host proteins interacting with dengue virus replication complex". Thesis, University of Bristol, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702423.

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Dengue virus (DENV) is a global health problem with approximately 390 million infections annually. Currently, no completely effective vaccines or medications are available to prevent DENV infection. This study used high-throughput quantitative proteomic analysis to identify cellular proteins that modulate DENV replication and are potential targets for the development of antiviral strategies. DENV is known to replicate and package its genome in association with perinuclear ER membranes. Initially, experimental conditions were optimised to infect human Huh-7 liver cells at high efficiency, with DENV serotypes -2 and -4 and to isolate a subcellular heavy membrane fraction (16K) enriched in the DENV replication complex. Then, stable isotope labelling by amino acids in cell culture (SILAC) coupled with high-throughput mass spectrometry (MS) was used to identify changes in the Huh-7 cell proteome and the subcellular 16K fraction in response to DENV-2 and DENV-4 infection. The analysis led to the identification and quantification of 3650 and 4026 and 3461 and 3668 proteins in the 16K and total cell proteomes from DENV-2 and DENV-4 infected cells respectively. For comparison, the total cell proteomes were also analysed by tandem mass tagging combined with MS. Proteomic bioinformatics was done on the datasets which showed that DENV modulated various cellular pathways including; protein biosynthesis, the secretory pathway, lipid metabolism and the cell cycle. A comparison of changes in the total and 16K proteomes from DENV infected cells identified a number of cellular proteins that selectively increased in the replicase containing fraction. which was validated by Western blotting and immunofluorescence analysis. Seven proteins (APOB, ARFRP1, BAG2, GOLGA1, GOLGB1, GOSRI and TMED9) were further investigated for their role in DENV -2 replication by examining either viral or replicon replication in cells depleted of the proteins by siRNA knockdown which revealed proteins that both positively and negatively modulated DENV replication.

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7

Griaud, François. "Proteomic analysis of leukaemogenic protein tyrosine kinase action". Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/proteomic-analysis-of-leukaemogenic-protein-tyrosine-kinase-action(ff9d490b-5a94-45fc-a857-4f0826e4a11a).html.

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Introduction: Chronic myeloid leukaemia is a blood cancer which progresses from a chronic phase to an acute blast crisis if untreated. Disease progression and treatment resistance may be precipitated by the mutator action of BCR/ABL protein tyrosine kinase (PTK), but only few protein phosphosites involved in the DNA damage response have been investigated with respect to BCR/ABL action. Aim: The aim of this PhD project was to demonstrate that BCR/ABL PTK expression can affect the response to genotoxic stress signalling at the protein phosphorylation level. Methodology: Etoposide-induced DNA damage response has been studied in control and BCR/ABL PTK-expressing Ba/F3 cells using apoptosis and γH2AX assays. Quantitative phosphoproteomics was performed with iTRAQ peptide labelling to discover putative modulated phosphorylation sites. Absolute quantification (AQUA ) performed with selected reaction monitoring was used to validate discovery phosphoproteomics. The effect of genotoxic stress on the THO complex protein Thoc5/Fmip was studied using western blots. Results: The expression of BCR/ABL PTK induced γH2AX phosphorylation after etoposide exposure. This was associated with the modulation of H2AX tyrosine 142 phosphorylation, MDC1 (serines 595 and 1053) and Hemogen serine 380 phosphorylation among proteins regulated by both BCR/ABL PTK and etoposide. We identified that leukaemogenic PTKs mediate Thoc5/Fmip phosphorylation on tyrosine 225 via Src proto-oncogene and oxidative stress, while ATM and MEK1/2 may control its phosphorylation. Human CD34+ CD38- leukaemic stem cells showed pronounced level of THOC5/FMIP tyrosine phosphorylation. Expression of phosphomutant Thoc5/Fmip Y225F might reduce apoptosis mediated by etoposide and H2O2. Conclusion: BCR/ABL PTK can sustain, create, block and change the intensity of protein phosphorylation related to genotoxic stress. Modulation of H2AX, MDC1, Hemogen and Thoc5/Fmip post-translational modifications by BCR/ABL PTK might promote unfaithful DNA repair, genomic instability, anti-apoptotic signalling or abnormal cell differentiation, resulting in leukaemia progression.

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8

Gilmore, Ian Richard. "Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells". Thesis, Swansea University, 2013. https://cronfa.swan.ac.uk/Record/cronfa42712.

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Oxysterols are oxygenated derivatives of cholesterol or its precursors. One oxysterol, 24(S),25-epoxycholesterol (24(S),25-EC), which results from a shunt in the cholesterol synthesis pathway has been found at higher than expected levels in embryonic murine brain. Interestingly, the receptor that 24(5),25-EC is a ligand for, Liver X Receptor (LXR), has been implicated in neurogenesis in the ventral mid brain region of embryonic brain; an area with a high density of dopaminergic neurons. The mechanism by which LXR induces this effect is unclear. Therefore, proteomic and phosphoproteomic studies were performed using a stable isotope labelled in amino acid in cell culture (SILAC) approach in order to quantify changes in the proteome between different treatment groups in a mouse substantia nigra dopaminergic cell line (SN4741) SN4741 cells were cultured in SILAC media containing differentially isotope labelled arginine and lysine. For protein expression studies SN4741 cells were treated in serum free media with vehicle, 10muM 24(S),25-EC, or 1muM GW3965, a synthetic ligand of LXR, for 24 hours. For analysis of changes in the phosphoproteome SN4741 cells were treated in serum free media with vehicle, 10muM 24(5),25-EC, or 30muM 25- hydroxycholesterol for 6 hours. Cells were lysed and protein combined in a 1:1 ratio before trypsin digestion and peptide separation via strong cation exchange chromatography. Phosphopeptides were enriched using immobilised metal affinity chromatography (IMAC). Resulting fractions were analysed, using a data dependent LC-MS/MS method. Data was quantified using MaxQuant software in conjunction with Mascot using an IPl mouse database. In protein expression analysis known oxysterol regulated genes, via SREBP or LXR, were differentially expressed. Oxysterol treatment induced global changes in proteins involved in lipid (cholesterol, fatty acid, phospholipid, triglyceride) synthesis. LXR? protein expression increased after GW3965 and 24(5),25-EC treatment, though no change was seen on LXRp mRNA, implying that ligand binding protects LXR? from degradation. 24(S),25-EC induced changes in expression and localisation of the membrane protein caveolin-1. Also, phosphoethanolamine cytidylyltransferase and collagen type IV alpha-3-binding protein, 2 proteins involved in phospholipid synthesis, had an altered expression after 24(S),25-EC treatment suggesting a role for oxysterols in membrane homeostasis. A cytokine, macrophage colony stimulating factor, which is required for normal neuronal development and macrophage differentiation had an LXR independent increased expression after 24(S),25-EC treatment. Quantitative RT-PCR data demonstrated that proteomic changes were due to both transcriptional and post-transcriptional effects of oxysterol. In addition, studies examining changes in the mouse phosphoproteome identified a number of novel phosphorylation sites.

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9

Puchalska, Monika [Verfasser] y Gerhard [Akademischer Betreuer] Mittler. "Quantitative proteomic analysis of the interactome of mammalian S/MAR (scaffold/matrix attachment region) elements". Freiburg : Universität, 2018. http://d-nb.info/1216826447/34.

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10

Saito-Benz, Hideshiro. "Identification of therapeutic targets to revert tamoxifen resistance by quantitative proteomic analysis of signaling networks". Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/61231.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, June 2009.
"April 2009." Cataloged from PDF version of thesis.
Includes bibliographical references.
Tamoxifen resistance is the biggest problem in endocrine treatment against hormone receptor positive breast cancer patients. HER2 is a membrane receptor tyrosine kinase that is known to correlate with poor disease outcome and unresponsiveness to endocrine treatment. Although much work has been done over the past decades to elucidate pathways involved in HER2 receptor signaling, the map of network-wide signaling events that contributes to the resistance to Tamoxifen treatment has not been characterized, making it difficult to pin-point the downstream drug target to revert the Tamoxifen resistance. To gain a molecular understanding of the mechanisms by which cells gain drug resistance, we have employed a proteomic analysis by mass spectrometry to quantitatively analyze cellular tyrosine phosphorylation signaling events in breast cancer model systems and human tumor samples. As a result of research, we have identified the major differences in downstream signaling pathways between Tamoxifen sensitive and Tamoxifen resistant breast cancer cell line models. These findings were further analyzed in Tamoxifen sensitive, and Tamoxifen treated/recurred patient samples to study clinical relevance. Specifically, we determined that P13K/Akt, MEK/ERK, and Src/FAK/Abl pathways are major components of the Tamoxifen resistance. We further showed that they signaling components are possible drug targets to revert Tamoxifen resistance. This study revealed cell-context specific network-wide changes in signaling events in response to use of therapeutic drugs. This is, to our first knowledge, the first phosphoproteomic analysis of the signaling network in breast cancer to address Tamoxifen resistance. We believe that same approach is applicable to other drug resistance problems in various disease settings.
by Hideshiro Saito-Benz.
Ph.D.

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11

Al, Zilay Abdulrhman. "High-throughput quantitative proteomic analysis of dengue virus infected THP-1 and K562 myeloid cell lines". Thesis, University of Bristol, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702903.

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12

Bertaccini, Diego. "Advances in analytical methodologies for the characterization and quantification in proteomic analysis". Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF043/document.

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L’objectif de cette thèse était de développer et d’optimiser de nouvelles méthodologies et approches analytiques afin d’améliorer le potentiel de l’analyse protéomique pour les études biologiques.La première partie de ce travail est consacrée à la détermination massive et exacte de la position N-Terminale des protéines (N-Terminome). Pour cela, nous avons utilisé et développé une approche basée sur une dérivation N-Terminale au TMPP. Cette méthodologie de marquage de la position N-Terminale a permis d’aborder l’étude des clivages protéolytiques des protéines exportées par le parasite P. falciparum (pathogène de la malaria) dans le globule rouge.Afin de permettre une exploitation automatique à haut débit des données de MS/MS, nous avons élaboré une nouvelle méthodologie (dénommée dN-TOP). Celle-Ci repose sur l’utilisation de TMPP portant des isotopes stables et permet ainsi d’accéder à la détermination des positions N-Terminales pour des études de N-Terminome à large échelle.La seconde partie est dédiée aux développements de différentes stratégies analytiques de quantification, aussi bien au niveau peptidique qu’au niveau protéique, appliquées à une série de problématiques biologiques. Ces optimisations ont été réalisées dans le contexte de l’étude des complexes protéiques, du dosage de prion par SRM, de quantification des glycations d’anticorps monoclonaux thérapeutiques et de l’hémoglobine HbA2 pour la standardisation des méthodes de référence
The objective of this Ph.D. thesis was to develop and optimize new methodologies and analytical approaches to improve the potential of the mass spectrometry based proteomics.The first part of this work focused on the development of the N-Termini proteomics. This topic was addressed with a specific N-Termini chemical derivatization based on TMPP. We have shown that our method allowed both specific N-Terminomics and classical proteomics studies in the same experiment.This N-Terminus methodology was applied to study the proteolytic cleavages of the exported proteins in P. falciparum, a parasite responsible for the malaria.In order to automatize the complex and tedious informatics processsing of the MS/SM data of ourTMPP based N-Terminomics method, we have introduced a new approach (named dN-TOP), based on the use of a stable isotope labeled TMPP which made now N-Terminome proteomics compatible with high throughput studies.The second part addresses quantitative aspects of proteomics. It describes the optimization of quantitative methods at the peptide level or at the protein level for five different proteomic studies in the context of protein complex subunits, targeted SRM based prion, quantification of monoclonal antibodies glycation and hemoglobin HbA2 for reference measurement methods standardization

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13

El, kennani Sara. "Investigation translationnelle de la chromatine Systematic quantitative analysis of H2A and H2B variants by targeted proteomics MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones". Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV059.

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L’ADN s’organise via les histones, les principaux acteurs dans la compaction du matériel génétique. L’évolution technologique a favorisé la découverte de nombreux variants protéique. Toutefois, l’annotation de ces derniers s’est faite de manière non conventionnelle, compliquant leur identification par spectrométrie de masse. Ainsi, j’ai développé une banque de données exhaustive, intitulée MS_HistoneDB, dédiée à la détection des variants d’histone par spectrométrie de masse. MS_HistoneDB permet l’utilisation d’échantillons murin et humain. En outre, l’utilisation de tests immunologiques permet difficilement de discriminer au niveau protéique des variants quasi-similaire. Ainsi, j’ai développé une méthode d’analyse de spectrométrie de masse ciblée pour détecter et quantifier les variants d’histones en un essai multiplexe. Cette méthodologie a été appliqué dans l’investigation de la chromatine au cours de la spermatogenèse, dans des modèles murins, physiologique ou pathologique mimant l’infertilité masculine.Un autre aspect de mon travail s’est intéressé aux protéines liant les formes modifiées des histones. Ainsi, j’ai étudié les « readers »de la famille BET (Bromodomaine et Extra-terminal domain). Ces protéines sont recrutées sur la chromatine via leurs bromodomaines, module spécifique reconnaissant les histones acétylées. Leur domaine extra terminal joue le rôle de plateforme de recrutement de régulateurs transcriptionnels. Ces protéines sont conservées chez la levure Saccharomyces cerevisiae,et également chez les pathogènes fongiques responsables d’infections invasives, où l’unique membre est appelé Bdf1. Ainsi, j’ai étudié le domaine extra-terminale de Bdf1 et démontré qu’il est essentiel à la survie des levures. Puis, j’ai exploré les mécanismes moléculaires impliqués. Enfin, des inhibiteurs sélectifs sont en cours de développement dans des espèces de levure pathogène. L’ensemble de ces travaux ouvrent la voie au développement d’une nouvelle classe thérapeutique d’antifongiques
DNA is organized via histones, the leading players in the compaction of genetic material. Technological evolution has favored the discovery of many protein variants. However, the annotation of the latter was unconventional, complicating their identification by mass spectrometry. Thus, I developed a comprehensive database, called MS_HistoneDB, dedicated to the detection of histone variants by mass spectrometry. MS_HistoneDB allows the use of murine and human samples. Also, the use of immunological tests makes it difficult to discriminate at the protein level of almost similar variants. Thus, I developed a targeted mass spectrometry analysis method to detect and quantify histone variants in a multiplex assay. This methodology has been applied in the investigation of chromatin during spermatogenesis, in mouse models, physiological or pathological mimicking male infertility.Another aspect of my work focused on proteins that bind modified forms of histones. Thus, I studied the "readers" of the BET family (Bromodomain and Extra-terminal domain). These proteins are recruited on chromatin via their bromodomains, a specific module recognizing acetylated histones. Their extra-terminal domain acts as a recruitment platform for transcriptional regulators. These proteins are conserved in the yeast Saccharomyces cerevisiae, and also in fungal pathogens responsible for invasive infections, where the only member is called Bdf1. Thus, I studied the extra-terminal domain of Bdf1 and demonstrated that it is essential for yeast survival. Then, I explored the molecular mechanisms involved. Finally, selective inhibitors are being developed in pathogenic yeast species. All of this work paves the way for the development of a new therapeutic class of antifungals

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14

Schulz, Melanie. "Quantitative proteomic analysis of early phosphorylation changes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in 5L rat hepatoma cells". Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-146738.

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15

Hawke, David H. "Quantitative mass spectrometry: Proteomic analysis of differentiation of MEL cells treated with hexamethylene bisacetamide and 7-[N-(3-aminopropyl)amino] heptan-2-one". Scholarly Commons, 2004. https://scholarlycommons.pacific.edu/uop_etds/2691.

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Polyamines are small, polycationic molecules required for growth and development and found in all living cells. In this study, the effects of two polyamine analogues, hexamethylene bisacetamide (HMBA), a differentiation inducer, and 7-[N-(3-aminopropyl)amino] heptan-2-one (APAH), an inhibitor of N8-acetylspermidine deacetylase, were studied using quantitative proteomics and stable-isotopes. Two new technologies, isotope-coded affinity tags (ICAT) and quantification in fragment spectra using isobaric stable isotope reagents (iTRAQ) were employed and compared. Quantitative results of these experiments showed few changes in the type and level of proteins detected in whole-cell extracts. Proteins from three populations of cells were studied, control (untreated), HMBA-treated, and HMBA plus APAH treated cells. Some of the proteins that were differentially expressed in response to these agents include pyruvate kinase (PK), lactate dehydrogenase (LDH), mini-chromosome maintenance protein 3 (MCM3), and poly-rC binding protein. The proteins PK and LDH have been reported as possible cancer markers. Histone protein levels were significantly reduced on HMBA treatment, and substantially recovered with the addition of APAH. This finding was very convincing in the iTRAQ work, but invisible to the ICAT experiment, because of the lack of cysteine residues required for quantification in the ICAT methodology. Two proteins were elevated in the HMBA-APAH experiment compared to the other two, heterogeneous nuclear ribonuclear protein C1/C2 (HNRP C1/C2) and ubiquitin. Considering their unique functions, the up-regulation of these proteins suggests the involvement of internal ribosome entry and protein degradation in response to APAH. The results of the two technologies, ICAT and iTRAQ, were found to overlap, but were partly complementary.

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16

Schulz, Melanie [Verfasser] y Friederike [Akademischer Betreuer] Eckardt-Schupp. "Quantitative proteomic analysis of early phosphorylation changes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in 5L rat hepatoma cells / Melanie Schulz. Betreuer: Friederike Eckardt-Schupp". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2011. http://d-nb.info/1025047222/34.

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17

Muller, Leslie. "Développements de méthodes de préparation d’échantillons pour l’analyse protéomique quantitative : application à la recherche de biomarqueurs de pathologies". Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF067/document.

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Les stratégies de protéomique quantitative sans marquage sont très attractives dans le domaine de la recherche de biomarqueurs de pathologies. Cependant, elles requièrent une pleine maîtrise du schéma analytique et de sa répétabilité. Plus particulièrement, la préparation d’échantillons nécessite d’être suffisamment répétable pour ne pas impacter la qualité et la fiabilité des résultats. Les objectifs de cette thèse étaient de développer et d’optimiser des méthodes analytiques pour la protéomique quantitative, en particulier pour l’étape de préparation d’échantillons. Ainsi, un protocole innovant, simple, rapide et permettant l’analyse quantitative sans marquage d’un grand nombre d’échantillons avec une haute répétabilité a été développé et optimisé : le « Tube-Gel ». Par ailleurs, des préparations d’échantillons adaptées à différentes matrices biologiques pour la recherche de biomarqueurs ont été élaborées. Les méthodes mises au point et leur application ont permis de proposer des candidats biomarqueurs pour plusieurs pathologies : le glioblastome, les lymphomes B diffus à grandes cellules, et les complications survenant sur les greffons rénaux
Label-free quantitative proteomics strategies are very attractive for diseases biomarkers researches. These approaches require the full control and the repeatability of the analytical workflow. In particular, the sample preparation has to be repeatable enough to ensure the quality and reliability of the results. Objectives of this work were to optimize and develop analytical methods for quantitative proteomics, with a special focus on the sample preparation step. Thus, an innovative, easy and fast protocol allowing the analysis of high sample numbers with high repeatability was developed and further optimized: the “Tube-Gel” protocol. Besides,sample preparations adapted to a variety of biological matrices were developed for the search of biomarkers. The developed methods and their application allowed the identification of potential biomarkers for a variety of diseases: glioblastoma, diffuse large B-cell lymphomas and renal transplants failures

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18

Takagi, Toshiyuki. "Studies on breeding of yeast Saccharomyces cerevisiae for effective macroalgae utilization based on the metabolism of marine bacterium". 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225665.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第20440号
農博第2225号
新制||農||1049(附属図書館)
学位論文||H29||N5061(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授植田充美, 教授小川順, 教授渡邊隆司
学位規則第4条第1項該当

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19

Walther, Dirk Martin. "Analysis of aging by quantitative proteomics and mitochondrial organellar proteomics". Diss., Ludwig-Maximilians-Universität München, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-174637.

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20

Stanislas, Thomas. "Rôle de la Dynamique Membranaire dans la Mise en Place des Mécanismes de Défense chez le Tabac". Phd thesis, Université de Bourgogne, 2011. http://tel.archives-ouvertes.fr/tel-00800206.

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La cryptogéine, une protéine sécrétée par l'oomycète Phytophthora cryptogea, provoque la mise en place de mécanismes de défense chez le tabac, mobilisant au cours des étapes précoces de la signalisation associée, des protéines localisées dans la membrane plasmique (MP). Une fraction membranaire résistante à la solubilisation par les détergents (DIM pour Detergent Insoluble Membrane), enrichie en stérols et en sphingolipides avait été purifiée à partir de la MP de tabac : cette fraction contenait plusieurs protéines impliquées dans la cascade de signalisation induite par la cryptogéine. Chez l'animal, l'association dynamique de protéines à des microdomaines riches en stérols et sphingolipides en réponse à un stress biotique joue un rôle essentiel dans la régulation de la signalisation cellulaire. L'objectif de ce travail était de tester l'hypothèse qu'un tel phénomène puisse se produire dans notre modèle d'étude. La comparaison du protéome de fractions DIMs, purifiées à partir de cellules traitées ou non pendant 5 minutes à la cryptogéine a été réalisée à l'aide d'un marquage isotopique (15N ou 14N) et d'une approche de protéomique quantitative. Le premier résultat est que l'abondance de la majorité des protéines n'est pas modifiée dans les DIMs en réponse à la cryptogéine. Une seule protéine est enrichie dans les DIMs, une isoforme de 14-3-3, tandis que quatre dynamines (DRPs pour Dynamin Related Proteins), impliquées dans le trafic vésiculaire, sont exclues des DIMs en réponse à la cryptogéine. L'étude d'une des dynamines identifiées, DRP1A, a été menée. Nous avons caractérisé les différents gènes codant DRP1A dans le génome du tabac, puis utilisé une approche ARN antisens pour altérer l'expression de cette protéine et nous avons étudié sa localisation subcellulaire à l'aide d'anticorps spécifiques et en observant en microscopie confocale cette protéine fusionnée à la GFP. Cette approche a permis de confirmer la présence de DRP1A dans la fraction DIMs et la diminution transitoire de son association à cette fraction en réponse à la cryptogéine, suite à une dissociation de la fraction membranaire. Ces travaux constituent la première mise en évidence d'une association/dissociation dynamique de protéines aux DIMs de plantes en réponse à un stimulus biologique

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21

Yang, Xu. "Quantitative Approaches for Protein Differential Expression Analysis". Thesis, Virginia Tech, 2009. http://hdl.handle.net/10919/36172.

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In this work, tandem mass spectrometry (MS/MS)-based quantitative protocols were developed to facilitate differential protein expression analysis and biomarker discovery via a two-step sample interrogation strategy: (a) global protein profiling and differential expression analysis by spectral counting; and, (b) biomarker candidate validation by targeted screening, i.e., multiple reaction monitoring (MRM). Preliminary experiments were performed to evaluate the performance of the spectral counting method. The method proved to be applicable for proteins with spectral countsâ ¥2, and a close-to-linear relationship between protein concentration and spectral count data was achievable at protein concentration levels <0.1 Î¼M. The detection limit was 40-800 fmol. A protein/peptide library containing ~10,000 peptide entries that facilitates the development of future MRM experiments, was developed. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum, the peptide retention time, and the top 10 most intense product ions that correspond to a given parent peptide. Only proteins identified by at least two spectral counts are listed. An MRM experiment was performed to demonstrate the successful applicability of this peptide library for the identification of putative biomarkers in proteomic samples.
Master of Science

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22

Anthony, Joseph Stephan. "A quantitative proteomics analysis of human cells undergoing apoptosis". Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/24167.

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Elucidating the events and mechanism of regulation of apoptosis is of wide interest to the scientific community, and to humanity, since apoptosis, so important for proper development and maintenance of an organism, is also responsible for disease when the process goes awry. In this thesis, a proteomics investigation into changes in protein concentrations and half-lives in early apoptosis is presented, enhancing our understanding of this process. Using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), cytokine withdrawal-induced apoptosis of a human hematopoietic cell line, TF-1, was studied. This is a useful model in which signaling pathways regulating apoptosis have been extensively studied previously. A study such as this can be considered “hypothesis-generating”, but at the outset the hypothesis is that proteins whose functions are closely tied to regulation of apoptosis will show detectable changes in quantity in cells undergoing apoptosis. Initially three biological replicates were performed, comprising 200 samples in all, analyzed using an FT-ICR mass spectrometer. Relative abundance of 1451 proteins identified in common between three biological replicates was determined, and 124 proteins showing the largest concentration changes in response to cytokine withdrawal are discussed in more detail. A subsequent effort investigated protein half-life changes in response to cytokine withdrawal and identified 255 proteins for which half-lives were calculated. The apparent changes in protein half-life in response to cytokine withdrawal are discussed. A high level of coverage of the proteome was achieved, giving a large number of protein identifications and relative quantitations. Further I have been able to identify several apparently synchronous changes in concentration between proteins with related functions, suggesting possible interactions not previously described, or identified as playing a role in cell survival, proliferation or death. Further, I observed cytokine withdrawal-induced alterations in concentration in some proteins for which little is known. The proteomic analysis of apoptosis using SILAC to determine protein half-life data is also a novel approach. Together, the work in this thesis suggests numerous avenues of investigation potentially leading to novel findings regarding cells undergoing apoptosis; and also suggests a potentially fruitful avenue of investigation for clinical management of patients undergoing chemotherapy.

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23

Frohn, Anne. "Structural and quantitative proteomic analyses of argonaute2-containing ribonucleoprotein complexes". Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-150792.

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24

Konstantinidis, Konstantinos. "Genome-Wide Proteomics and Quantitative Analyses on Halophilic Archaea". Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-101048.

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25

Fisher, Christal. "Quantitative analysis of the plasma proteome in pre-eclampsia". Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/quantitative-analysis-of-the-plasma-proteome-in-preeclampsia(3e207341-ebb9-4cb0-b7ea-34b9b110eda6).html.

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There is currently no clinically useful screening test available to identify nulliparous women at high risk of developing pre-eclampsia. This study aimed to identify novel biomarkers using hypothesis generating proteomic methods applied to plasma samples obtained prior to clinical diagnosis of pre-eclampsia. Plasma samples taken at 15 weeks gestation from women who subsequently developed late pre-eclampsia (> 34 weeks), early pre-eclampsia (< 34 weeks) and two distinct groups of women with uncomplicated pregnancies (each n=12) were pooled. Pooled plasma was immunodepleted, labelled using iTRAQ-8 plex reagent and separated into fractions using high pH reverse phase chromatography. Fractions were analysed by LC-MS/MS and data interrogated using ProteinPilot 3.0. The merits of two immunodepletion systems were compared; the Seppro® IgY 14 -SuperMix LC column system removes up to 100 highly abundant plasma proteins and the Multiple Affinity Removal LC column depletes 14 highly abundant plasma proteins. Removal of more high abundance proteins allowed identification of more, potentially interesting, low abundance proteins, but was less reproducible than removing fewer proteins. Two methods of LC-MS/MS analysis were assessed; the QStar XL qTOF and 5800 MALDI-TOF-TOF. The protein identifications and the quantification data acquired by each method was comparable and complementary and increased the total number of proteins identified. A total of 502 proteins were identified. A stringent two stage analysis was developed to identify candidate proteins which changed in abundance in plasma from women who later developed pre-eclampsia compared to women with uncomplicated pregnancies. Analysis identified a total of 113 proteins which were both reproducibly quantified and changed by more than the expected range of biological variation. Six candidate proteins changed in abundance in the plasma taken from women who subsequently developed early pre-eclampsia were selected for further validation. A high throughput, low cost, method of multiple reaction monitoring which allows relative quantitation without the use of costly isotopically labelled peptides was developed to validate candidate proteins. Candidate proteins were also assessed by western blot and ELISA. Only one candidate protein; platelet basic protein, was validated by all three methods and demonstrated similar increases in the abundance. This investigation suggests that measurement of platelet basic protein at 15 weeks gestation is a novel candidate predictive marker for pre-eclampsia. Validation of platelet basic protein in a large, independent, sample set is required to confirm changes in protein expression and to evaluate potential, alongside other factors, to identify nulliparous women at high risk of developing pre-eclampsia later in pregnancy.

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26

Kristensen, Anders Riis. "Analysis of the regulation of biological networks using quantitative proteomics". Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44813.

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A protein network can be thought of as a graph with nodes and edges, where nodes represent proteins and edges represent protein-protein interactions. Neither proteins nor their interactions are stable constituents in the cell; they are constantly changing in response to external stimulation or internal programming. Changes in protein expression are regulated by transcription, translation and protein degradation, whereas protein interaction changes have been shown in focused studies to be regulated by post translational modification. To investigate the processes influencing the regulation of protein expression and interaction changes, mass spectrometry based proteomics was applied because it has two key advantages for the study of protein networks: 1) it directly detects peptides from the proteins, and so does not rely on antibodies or the generation of fusion proteins; 2) by combining mass spectrometry based proteomics with quantitative techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), it is possible to quantify thousands of proteins in a single experiment. Here a systems biology approach was applied to investigate protein expression change, synthesis and degradation of proteins during cellular differentiation in two different cell lines. This allowed observing that protein expression during cellular differentiation is largely controlled by changes in the relative synthesis rate, whereas the relative degradation rate of the majority of proteins changed little. By comparing the data with previously published data of mRNA levels, there could be provide strong evidence that the generally poor correlation observed between transcript and protein levels can be explained once the protein synthesis and degradation rates are taken into account. To study how protein interactions change in response to perturbation, a novel approach was developed combining size exclusion chromatography (SEC) and protein correlation profiling (PCP)-SILAC. Stimulation with epidermal growth factor (EGF) caused 351 proteins to alter their interactions with other proteins and, interestingly, when compared to previously published phosphorylation data, these proteins tended to also have altered phosphorylation under similar experimental conditions. This approach allowed identification of protein interactions in numbers comparable to other high throughput techniques, but also enabled quantification of protein stoichiometry between proteins participating in multiple complexes.

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27

Ahmad, Yasmeen. "Management, visualisation & mining of quantitative proteomics data". Thesis, University of Dundee, 2012. https://discovery.dundee.ac.uk/en/studentTheses/6ed071fc-e43b-410c-898d-50529dc298ce.

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Exponential data growth in life sciences demands cross discipline work that brings together computing and life sciences in a usable manner that can enhance knowledge and understanding in both fields. High throughput approaches, advances in instrumentation and overall complexity of mass spectrometry data have made it impossible for researchers to manually analyse data using existing market tools. By applying a user-centred approach to effectively capture domain knowledge and experience of biologists, this thesis has bridged the gap between computation and biology through software, PepTracker (http://www.peptracker.com). This software provides a framework for the systematic detection and analysis of proteins that can be correlated with biological properties to expand the functional annotation of the genome. The tools created in this study aim to place analysis capabilities back in the hands of biologists, who are expert in evaluating their data. Another major advantage of the PepTracker suite is the implementation of a data warehouse, which manages and collates highly annotated experimental data from numerous experiments carried out by many researchers. This repository captures the collective experience of a laboratory, which can be accessed via user-friendly interfaces. Rather than viewing datasets as isolated components, this thesis explores the potential that can be gained from collating datasets in a “super-experiment” ideology, leading to formation of broad ranging questions and promoting biology driven lines of questioning. This has been uniquely implemented by integrating tools and techniques from the field of Business Intelligence with Life Sciences and successfully shown to aid in the analysis of proteomic interaction experiments. Having conquered a means of documenting a static proteomics snapshot of cells, the proteomics field is progressing towards understanding the extremely complex nature of cell dynamics. PepTracker facilitates this by providing the means to gather and analyse many protein properties to generate new biological insight, as demonstrated by the identification of novel protein isoforms.

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28

Walther, Dirk Martin [Verfasser] y Matthias [Akademischer Betreuer] Mann. "Analysis of aging by quantitative proteomics and mitochondrial organellar proteomics / Dirk Martin Walther. Betreuer: Matthias Mann". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1058822098/34.

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29

Mackay, Katherine. "A comparative study of analysis methods in quantitative label free proteomics". Thesis, University of Liverpool, 2015. http://livrepository.liverpool.ac.uk/2050359/.

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The large amounts of data generated by modern proteomics experiments necessitates the use of software pipelines to conduct the bulk of the post-processing. While many software packages (both commercial and open-source) are available to perform some or all of the necessary post-processing steps, it is usual for each research group to use only the instrumentation and software packages with which they are most familiar and/or which are available to analyse their unknown data. The intention of the studies presented within this thesis was to assess the correlation between the experimental results obtained when; - a single result dataset is obtained and post-processed in parallel using four separate software pipelines - a single sample is analysed on two different mass spectrometers and post-processed in parallel and; - when different identification thresholds are applied to a dataset prior to parallel quantitation of the resultant data sets Correlation between different mass spectrometry instruments was assessed and found to yield high r values, especially at the protein level, and was also found to improve following the application of abundance thresholds, however the result of applying score thresholds was unpredictable. The use of manual FDR thresholds prior to importing data into Progenesis LC-MS yielded interesting results, which suggest that a threshold of 1% peptide FDR and 1 or 2% protein FDR is most effective in terms of yielding accurate ratios while maintaining acceptable sensitivity. In addition, a consensus method is suggested to utilise the results from multiple software pipelines in order to increase sensitivity and reduce the FDR, through the use of the QPROT tool and manual post-processing.

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30

Becker, René. "Quantitative Proteomanalyse des prädatorischen Bakteriums Bdellovibrio bacteriovorus". Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/19551.

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Durch den exzessiven Gebrauch von Antibiotika haben sich in den letzten Jahren zunehmend Resistenzen herausgebildet. Eine potentielle Alternative zu konventionellen Antibiotika sind prädatorische Bakterien. Das Bakterium Bdellovibrio bacteriovorus hat einen zweiphasigen Lebenszyklus bestehend aus einer Angriffsphase, in der es andere gram-negative Bakterien jagt, und einer Wachstumsphase, in der es das Zytoplasma eines Wirtes für die eigene Reproduktion nutzt. Für einen künftigen Einsatz von B. bacteriovorus als Antibiotikum müssen die Prozesse des Lebenszyklus verstanden werden. Das Proteom von B. bacteriovorus wurde bisher jedoch nur sehr wenig untersucht. Daher wurden in dieser Arbeit mithilfe der Massenspektrometrie Proteine von verschiedenen Zeitpunkten des Lebenszyklus von B. bacteriovorus relativ quantifiziert. Es konnten zahlreiche Proteine identifiziert werden, die zu spezifischen Zeitpunkten des Lebenszyklus hoch- oder herabreguliert werden. Die größten Unterschiede im Proteinmuster konnten zwischen der Angriffs- und der Wachstumsphase beobachtet werden. In der Angriffsphase sind einige Proteine herabreguliert, die mit der Proteinexpression im Zusammenhang stehen. Weiterhin wurde bestätigt, dass sich junge und gealterte Zellen der Angriffsphase deutlich voneinander unterscheiden, womit die Angriffsphase eigentlich aus zwei Phasen besteht. Auf Grundlage der Ergebnisse und eines Vergleiches mit Transkriptionsdaten wurde die Vermutung aufgestellt, dass B. bacteriovorus Proteine, welche spezifisch für die Angriffsphase sind, bereits während der Wachstumsphase synthetisiert. Im Zusammenhang mit der Forschung an B. bacteriovorus konnten auch neue Impulse bezüglich der MeCAT-basierten massenspektrometrischen Proteinquantifizierung angestoßen werden. In dieser Arbeit wurde unter anderem ein MeCAT-Reagenz mit Acrylamidfunktionalität entwickelt, welches erfolgreich als interner Standard für die Laserablation-ICP-MS von Polyacrylamidgelen verwendet werden kann.
Due to the excessive use of antibiotics, antibiotic resistance has increased over the last years. A potential alternative to conventional antibiotics are predatory bacteria. The predatory bacterium Bdellovibrio bacteriovorus has a biphasic life cycle consisting of an attack phase in which it hunts other gram-negative bacteria, and a growth phase in which it uses the cytoplasm of a prey cell as a substrate for its own reproduction. For future application of B. bacteriovorus as an antibiotic, it is necessary to understand the processes that occur during the life cycle. However, almost no information has been obtained regarding the proteome of B. bacteriovorus yet. Using mass spectrometry and an isotopic labelling strategy, proteins from different time points in the life cycle of B. bacteriovorus were quantified relatively to each other in this work. Numerous proteins were identified that are up- or down-regulated at specific time points in the life cycle. The largest differences in protein pattern existed between the attack phase and the growth phase, whereas only minor differences occurred within the growth phase. For instance, several proteins that appear to be down-regulated during the attack phase are related to protein expression. Furthermore, it was confirmed that there is a significant difference between young and aged cells of the attack phase. Therefore, the attack phase actually consists of two phases. Based on the results and on a comparison with transcription data, it was suggested that attack phase specific proteins of B. bacteriovorus are already synthesized during the growth phase. In connection with the research on B. bacteriovorus, new impulses regarding the MeCAT based protein quantification with mass spectrometry could be initiated. In this work, a MeCAT reagent with acrylamide functionality was developed, which can be used successfully as an internal standard for laser ablation ICP-MS of polyacrylamide gels.

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31

Lau, Edward y 劉家明. "Combinatorial use of SCX and RP-RP separation for iTRAQ-based quantitative proteomics profiling". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44923120.

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32

Chen, Jiaxuan. "Analysis of protein-protein interaction by in vivo quantitative proteomics in Caenorhabditis elegans". Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17331.

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In C. elegans bietet die frühe Embryogenese ein attraktives Modellsystem, um Wechselwirkungen von Proteinen in vivo zu entschlüsseln. Zur präzisen Identifizierung von spezifischen Interaktionen im C. elegans Embryo wurde ein neuer quantitativer Ansatz entwickelt, welcher die Expression von Fusionsproteinen an grün fluoreszierendes Protein in vivo mit markierungsfreier Interaktionsproteomik kombiniert. Diese Strategie wurde angewandt, um die Interaktionspartner von acht Proteinen zu untersuchen, die in essentiellen biologischen Prozessen während der frühen Embryogenese involviert sind. Diese Studie liefert als Ergebnis ein erstes embryonales in vivo Interaktionsnetzwerk bestehend aus 559 Interaktionen zwischen 472 Proteinen. Dieses Netzwerk erfasst nicht nur bekannte Bindungen, sondern auch neue Interaktionen von hoher funktioneller Relevanz. Die Netzwerkinformationen wurden mit Experimenten auf Basis der Ribonukleinsäuren-Interferenz kombiniert um neue Regulatoren der sogenannten „P granules” ausfindig zu machen. Infolgedessen wurde das fadenwurmspezifische Protein GEI-12 als neuer Interaktionspartner der DYRK-Kinase MBK-2 und als wichtiger Regler für die Dynamik der „P granules“ und für die Aufrechterhaltung der Keimbahn identifiziert. Dies führt zu einem hypothetischen Modell in welchem der Phosphorylierungszustand von GEI-12 den Auf- und Abbau der „P granules“ während der frühen Embryogenese vermittelt. Darüber hinaus veranlasst GEI-12 auch die Entstehung von „P granules“ in Säugetierzellen und bindet an PP2A-Phosphatasen, was darauf hindeutet, dass die grundlegenden biophysikalischen Eigenschaften die zur Entstehung der Ribonukleoprotein-Körperchen notwendig sind, im Laufe der Evolution zwischen Spezies konserviert geblieben sind. Zusammenfassend stellt die in vivo Interaktionskartierung ein vielseitiges Werkzeug dar, welches nicht nur die funktionelle Organisation des Proteoms aufdeckt, sondern auch Einsichten in die tierische Entwicklungsbiologie liefert.
In C. elegans, early embryogenesis provides an attractive model system for mapping in vivo protein interactions. In order to accurately identify specific interactions in C. elegans embryos, a new quantitative approach was developed combining in vivo expressed GFP fusion proteins with label-free interaction proteomics. This strategy was applied to studying the interaction partners of eight bait proteins involved in essential biological processes during early embryogenesis. As a result, this study generated a pilot embryo in vivo interaction network composed of 559 interactions among 472 proteins. Importantly, this network captures not only well-characterized bindings but also new interactions of high functional relevance. Further utility of the network is demonstrated by combining it with RNAi perturbation to search for new regulators of P granule formation in early embryos. Consequently, a worm-specific protein GEI-12 was discovered as a novel interaction partner of the DYRK kinase MBK-2 and as an important regulator of P granule dynamics and germline maintenance. This leads to a hypothetical model in which the phosphorylation state of GEI-12 mediates P granule assembly and disassembly during early embryogenesis. In addition, GEI-12 also induces granule formation in mammalian cells and interacts with PP2A phosphatases, indicating that the fundamental biophysical properties required for ribonucleoprotein granule formation are conserved across species during evolution. In summary, in vivo interactome mapping is a versatile approach that not only unravels the functional organization of the proteome but also can reveal insights into animal development.

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33

Jarnuczak, Andrew. "Mass spectrometry-based quantitative proteomics applied to the analysis of Saccharomyces cerevisiae heat stress response and chaperone deletion strains". Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/mass-spectrometrybased-quantitative-proteomics-applied-to-the-analysis-of-saccharomyces-cerevisiae-heat-stress-response-and-chaperone-deletion-strains(c653915b-70fa-44d7-9bb7-6e7965349ff0).html.

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In the last decade omics technologies enabled detailed and system-wide analysis of complex biological samples. Genomics, transcriptomics and metabolomics all benefited tremendously from technological advances in their respective fields. Proteomics was revolutionised by mass spectrometry, which allowed simultaneous identification of thousands of proteins in cells, tissues and organisms. And this mainly qualitative revolution, quickly turned quantitative. This work had two main objectives. Firstly, to apply the state of the art instrumentation, data analysis and bioinformatics methods to better our understanding of basic cell biology in a model organism Saccharomyces cerevisiae. Specifically, to quantitatively describe the effects of perturbations, such as adverse environmental conditions or chaperone gene deletions, on protein abundances in the cell. Additionally, it was aimed to demonstrate and evaluate the ability of a new timeof-flight mass spectrometer to perform large-scale absolute quantification. First, it was found that yeast cells are remarkably robust to deletions of major chaperone hub proteins (Ssa1p or Ssb1p deletions). This ability was attributed to network structure and redistribution of folding workload among other related chaperones rather than simple functional redundancy. Second, to build on the first set of results, a detailed time resolved description of yeast proteome dynamics in response to heat stress was provided for the wild type and Ssb1p chaperone mutant strains. In this study, for the first time in the literature, temporal expression patterns of many hallmark heat shock proteins were elucidated. Globally, a slow and sustained proteome remodelling or 'buffering' was revealed in both strains. However, it was also shown that the cells knocked out for the Ssb1p chaperone respond to heat in a distinctly different manner to the wild type strain. Finally, consistent and reproducible absolute quantification of multiple yeast proteomes was demonstrated using a new commercial time-of-flight mass spectrometer with ion mobility separation capabilities. The data obtained revealed global differences in cellular protein content between various chaperone prefoldin mutants as well as differential expression of a set of proteins promising to be interesting targets for further investigations.

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34

Plumel, Marine. "Optimisations des stratégies analytiques quantitatives en protéomique : application à l'étude des réponses adaptatives du métabolisme chez divers organismes". Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF014/document.

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L’analyse protéomique consiste à caractériser l’ensemble des protéines exprimées dans une cellule, dans un état et à un temps donné (le protéome). L’analyse protéomique tend aujourd’hui à fournir des informations quantitatives, cependant, pour pouvoir générer des données fiables et sensibles, des optimisations méthodologiques doivent être apportées à chaque problématique biologique. L’objectif de cette thèse a été d’adapter les stratégies protéomiques quantitatives existantes à diverses problématiques biologiques. Ce travail de thèse a ainsi contribué à apporter des résultats originaux concernant l’étude de désordres métaboliques. Il a également permis la mise au point d’une méthode de suivi du statut reproducteur de la tortue Luth. Finalement, il a permis d’évaluer la réponse d’une lignée cellulaire hépatique à une intoxication chronique à l’acide valproique. Ces données alimenteront des algorithmes de prédiction de toxicité hépatique chronique au travers du consortium NOTOX (EU)
Proteomics consists in the characterization of all the proteins expressed in a physiological state and at a given time (the proteome). Today, proteomics tends to bring quantitative information. However, in order to generate reliable, sensitive and robust data, whatever the biological question, methodological improvements need to be done. The aim of this PhD thesis was to apply and adapt quantitative proteomics strategies to specific biological issues as there is no universal quantifying method. This PhD thesis contributed to bringing original results concerning the study of metabolic disorders. It has also contributed to setting-up a targeted approach in order to quantify the level of reproductive effort of Leatherback Turtles. Finally, this PhD thesis also contributed, through the consortium NOTOX (EU), to the evaluation of physiological answers of hepatic cell lines exposed to valproic acid. These data will be used in order to generate hepatotoxicity prediction algorithms

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35

Frohn, Anne [Verfasser] y Klaus [Akademischer Betreuer] Förstemann. "Structural and quantitative proteomic analyses of argonaute2-containing ribonucleoprotein complexes / Anne Frohn. Betreuer: Klaus Förstemann". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2011. http://d-nb.info/1028490496/34.

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36

Demant, Myriam. "Qualitative and quantitative proteome analyses of bovine oocytes and early embryos". Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-142446.

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37

Grady, Joshua Terrence Wilson. "Adapting Quantitative Protein and Phosphorylation Analyses to a Proteome-Wide Scale". Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10852.

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Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has become the preferred method for large-scale peptide and phosphopeptide identification and quantification. The dominance of LC-MS/MS is the result of improved chromatographic, mass spectrometry and bioinformatic technologies. The applications of these technological improvements drive biological innovation by expanding the realm of possible experimentation, facilitating the creation and evaluation of novel hypotheses. Such improvements are the focus of this dissertation. New technologies are presented and their proteome wide applications in biological systems are demonstrated. A comparison of common phosphopeptide enrichment methods is presented in chapter two, which demonstrates that a combination of methods provides non-overlapping data sets. This comparison was performed in mitotically arrested fission yeast, a previously unstudied system by phosphoproteomic methods. This chapter remarks upon phosphorylation site conservation between lower and higher eukaryotes, as a means of predicting potentially relevant phosphorylation events in mammals. A new protocol for tissue based peptide quantification is presented in chapter three. The large-scale application of this method is detailed in a system of mouse liver phosphorylation, between fasted and re-fed states. The effect of peptide and protein level false discovery rates on the accuracy of phosphorylation site quantification is highlighted. This method is a cost-effective alternative to available techniques, such as metabolic labeling, and expands the application of proteomics to include larger animals. Finally, an in depth analysis of quantitative LC-MS/MS based multiplexing is the subject of the last chapter. New techniques for peptide pre-fractionation and ion quantification are discussed, which improve proteome coverage and quantitative accuracy. This proteome-wide multiplexing is applied to an analysis of the budding yeast environmental stress response. Applicable methods of data processing and a means of obtaining biologically relevant information out of multidimensional proteomic data sets are discussed. In all chapters, the data presented represent the largest analyses of their kind. This dissertation provides a solid guide for future proteome-wide studies, focused on the identification and quantification of peptides and their posttranslational modifications.

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38

Müller, Stephan [Verfasser], Andrea [Akademischer Betreuer] Sinz, Sacha [Akademischer Betreuer] Baginsky y Martin [Akademischer Betreuer] Bergen. "Optimization of quantitative proteomic LC-MS analyses and proteomic insights into Helicobacter pylori : [kumulative Dissertation] / Stephan Müller. Betreuer: Andrea Sinz ; Sacha Baginsky ; Martin Bergen". Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2013. http://d-nb.info/1048047121/34.

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39

Mnatsakanyan, Ruzanna [Verfasser] y Oliver J. [Akademischer Betreuer] Schmitz. "Development and application of quantitative proteomics method for S-nitrosylation analysis / Mnatsakanyan Ruzanna ; Betreuer: Oliver J. Schmitz". Duisburg, 2021. http://d-nb.info/1230322604/34.

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Vaca, Jacome Alvaro Sebastian. "Progress towards a better proteome characterization by quantitative mass spectrometry method development and proteogenomics". Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAF020/document.

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L'extrême complexité des échantillons biologiques, la variabilité technique et la dépendance de la protéomique envers les banques protéiques empêchent l'analyse complète d'un protéome. Ce travail de thèse s'est focalisé sur le développement de méthodes pour la protéomique quantitative et la protéogénomique afin d'améliorer la caractérisation du protéome. Premièrement mon travail s'est centré sur le développement de méthodes quantitatives globales et ciblées. La mise en place de standards pour évaluer les performances de tous les niveaux de la stratégie analytique est aussi décrite. Ces méthodes ont été optimisées pour répondre à diverses questions biologiques. Mon doctorat s'est focalisé aussi autour de la protéogénomique. Une méthode d'analyse N-terminomique à haut débit a été développée et appliquée à l'étude de la mitochondrie humaine. Enfin, ce manuscrit présente une approche multi-omique visant à améliorer l'analyse du protéome avec la création de banques de données personnalisées
The high intrinsic complexity of biological samples, the technical variability and the dependency of Bottom-up Proteomics to consensus protein sequence databases handicap the comprehensive analysis of an entire Proteome. My doctoral work was focused on method development in quantitative Proteomics and Proteogenomics in order to achieve a better proteome characterization. First, I focused on the development of global and targeted quantitative methods. The introduction and development of standard samples to assess the performances at any level of the analytical workflow is also described. These methods were applied to answer different biological questions. My PhD also focused on Proteogenomic method development. A high throughput N-terminomic analysis approach was developed and applied to the analysis of the human mitochondria. Finally, this manuscript presents a personalized multi-omics profiling strategy to improve the proteome analysis with the use of personalized databases

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41

Wijasa, Teodora Stella [Verfasser]. "Quantitative proteome analysis of S-nitrosylation on synaptosomal proteins in Alzheimer's disease / Teodora Stella Wijasa". Bonn : Universitäts- und Landesbibliothek Bonn, 2019. http://d-nb.info/1198933828/34.

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42

Hubel, Philipp [Verfasser] y Matthias [Akademischer Betreuer] Mann. "Illuminating novel aspects in virus-host interactions by tailored quantitative proteomics analyses / Philipp Hubel ; Betreuer: Matthias Mann". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/121985221X/34.

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43

Demant, Myriam Verfasser] y Eckhard [Akademischer Betreuer] [Wolf. "Qualitative and quantitative proteome analyses of bovine oocytes and early embryos / Myriam Demant. Betreuer: Eckhard Wolf". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1022523457/34.

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44

Chen, Jiaxuan [Verfasser], Richard [Akademischer Betreuer] Lucius, Matthias [Akademischer Betreuer] Selbach y Markus [Akademischer Betreuer] Landthaler. "Analysis of protein-protein interaction by in vivo quantitative proteomics in Caenorhabditis elegans / Jiaxuan Chen. Gutachter: Richard Lucius ; Matthias Selbach ; Markus Landthaler". Berlin : Lebenswissenschaftliche Fakultät, 2015. http://d-nb.info/1078309507/34.

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45

El, Kashef Noha Hussein Mohamed Salama [Verfasser], Bent [Akademischer Betreuer] Schneider y Bent [Akademischer Betreuer] Brachvogel. "Proteome-based diagnostics of SIDS : comparative quantitative analysis / Noha Hussein Mohamed Salama El Kashef ; Akademische Betreuer: Bent Schneider, Bent Brachvogel". Köln : Deutsche Zentralbibliothek für Medizin, 2018. http://d-nb.info/1172078130/34.

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El-Kashef, Noha Hussein Mohamed Salama [Verfasser], Bent [Akademischer Betreuer] Schneider y Bent [Akademischer Betreuer] Brachvogel. "Proteome-based diagnostics of SIDS : comparative quantitative analysis / Noha Hussein Mohamed Salama El Kashef ; Akademische Betreuer: Bent Schneider, Bent Brachvogel". Köln : Deutsche Zentralbibliothek für Medizin, 2018. http://d-nb.info/1172078130/34.

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47

El-Kashef, Noha Hussein Mohamed Salama [Verfasser], Bent Akademischer Betreuer] Schneider y Bent [Akademischer Betreuer] [Brachvogel. "Proteome-based diagnostics of SIDS : comparative quantitative analysis / Noha Hussein Mohamed Salama El Kashef ; Akademische Betreuer: Bent Schneider, Bent Brachvogel". Köln : Deutsche Zentralbibliothek für Medizin, 2018. http://nbn-resolving.de/urn:nbn:de:hbz:38m-frl:64113847.

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48

Aburaya, Shunsuke. "Studies on molecular recognition and degradation mechanism of plant cell wall polysaccharides-assimilating Clostridium cellulovorans using proteome analysis". Kyoto University, 2019. http://hdl.handle.net/2433/242685.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21808号
農博第2321号
新制||農||1066(附属図書館)
学位論文||H31||N5180(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授植田充美, 教授渡邊隆司, 教授栗原達夫
学位規則第4条第1項該当

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49

Mappa, Charlotte. "Exploration de nouveaux concepts pour les analyses quantitatives et fonctionnelles de microbiotes modèles d'intérêt dual". Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT055.

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La détection et l’identification de micro-organismes pathogènes est un réel enjeu de santé public tant au niveau alimentaire, que clinique ou d’intérêt national tel qu’illustré en biodéfense. Dans tous ces domaines, il est important d‘avoir des méthodes d’identification et de détection qui soient à la fois rapide, sensible et robuste. Cette thèse a pour objectif de contribuer au développement d’une approche rapide d’identification de micro-organismes sans a priori par spectrométrie de masse en tandem. Cette approche innovante, appelée phylopeptidomique, repose sur l’alliance de la peptidomique, i.e. analyse à large échelle des peptides provenant de la digestion enzymatique d’un échantillon biologique, et de la phylogénie des organismes cellulaires. Après extraction des protéines présentes dans l’échantillon à ausculter, des peptides sont générés et analysés par spectrométrie de masse en tandem. La déconvolution des signaux MS/MS à l’aide du logiciel « µOrg.ID » développé en propre au laboratoire permet d’identifier et quantifier les organismes présents dans l’échantillon en fonction des organismes indexés dans les bases de données. L’étude du protéome de Bacillus atrophaeus, agent simulant de l’anthrax, sous forme sporulée et végétative a permis d’illustrer une méthode d’identification de biomarqueurs protéiques permettant de déterminer le ratio entre les deux formes dans un échantillon. La limite de détection de la phylopeptidomique dans des échantillons purs et des échantillons en mélange équimolaire a été établie sur des bactéries modèles d’intérêts médical et environnemental. La limite de détection de spores de Bacillus atrophaeus en présence de 14 matrices interférentes (alimentaires, environnementales et autres) a permis de mettre en évidence les avantages et limitations de l’approche. Enfin, un mélange artificiel standardisé de 24 organismes a été développé afin d’évaluer les outils de bio-informatique en métaprotéomique
The detection and identification of pathogenic microorganisms is a real public health issue for the food industry and the clinics or national interest as illustrated in the biodefense field. Thus, it is important to have identification and detection methods that are fast, sensitive and robust. This PhD thesis aims at contributing to the development of a rapid approach to identify microorganisms without any a priori by tandem mass spectrometry. This innovative approach, called phylopeptidomics, is based on the combination of peptidomics, i.e. large scale analysis of peptides derived from the enzymatic digestion of a biological sample, and the phylogeny of cellular organisms. After extraction of the proteins from the sample of interest, peptides are generated and analyzed by tandem mass spectrometry. The deconvolution of MS/MS signals using the "μOrg.ID" software developed in the laboratory enables the identification and quantification of organisms present in the sample according to the organisms indexed in generalist databases. The study of the proteome of Bacillus atrophaeus, a simulant agent of anthrax, in sporulated and vegetative form, has provided an illustration of a new method of identification of protein biomarkers, which allows determining the ratio between both forms. The limit of detection of phylopeptidomics in pure samples and equimolar mixtures was established with model bacteria of medical and environmental interests. The limit of detection of B. atrophaeus spores in the presence of 14 interfering matrices (food, environmental and others) has highlighted the advantages and limitations of the approach. Finally, a standardized artificial mixture of 24 organisms was developed in order to evaluate bioinformatics tools in metaproteomics

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50

Orton, Christopher R. "Analysis of Protein Adduction Kinetics and the Effects of Protein Adduction on C-Jun N-Terminal Kinase Signaling". Diss., The University of Arizona, 2006. http://hdl.handle.net/10150/194247.

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Defining the mechanics and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. In this dissertation I describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein using quantitative mass spectrometry. Adducts are formed by electrophiles at Cys-14 and Cys-47 on the metabolic enzyme glutathione-S-transferase P1-1 and accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples. This method was used to measure rate constants for adduction at both positions with two different model electrophiles, IAB and BMCC. The results indicate that Cys-47 was approximately 2-3-fold more reactive toward both electrophiles than was Cys-14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system. Quantitative analyses of protein modifications provide a means of determining the reactivity and selectivity of damaging protein modifications in chemical toxicity.Another area of study explored in this dissertation is looking at the effects of protein alkylation on activating cellular signaling pathways, specifically the JNK signaling pathway. Protein adduction has been shown to be selective between different alkylating agents. It would then be reasonable to think this selectivity of adduction translates to selectivity of downstream consequences or cellular events directly tied to specific adductions. My work will show how treatment of HEK293 cells with either IAB or BMCC leads to differences in activation of JNK signaling. In addition, I've been able to show a difference in selectivity of a number of adducted targets by each alkylating agent, which are directly involved in regulation of the JNK signaling pathway. These studies illustrate not only the significance of protein adduction, but the importance for continual research to better understand their behavior in living systems.

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