O metabolismo celular é substancialmente alterado durante a oncogênese, progressão tumoral e outros processos patológicos. Tem sido frequentemente analisado para compreensão dos processos que permitem o crescimento dos tecidos, reprodução, manutenção da homeostase e resposta a sinais extracelulares. Dentre os vários métodos para caracterização de alterações metabólicas, a espectrometria de massas tem contribuído significativamente para a identificação e quantificação de proteínas envolvidas no metabolismo, e também para a caracterização do metaboloma. A análise proteômica baseada em espectrometria de massas permite estudos qualitativos em grande escala, adequados para a busca e descoberta de analitos relevantes. A análise proteômica dirigida complementa esse caráter com qualidade quantitativa para proteínas alvo pré-selecionadas. Neste trabalho foram desenvolvidos métodos de proteômica dirigida para o monitoramento de alterações quantitativas nos níveis de proteínas envolvidas na via glicolítica do metabolismo da glicólise. Para tal peptídeos proteotípicos para cada proteína foram identificados e padronizados utilizando a estratégia de monitoramento de reações múltiplas. O painel foi aplicado para obter resultados das alterações que ocorrem em um modelo de progressão tumoral. Com esta estratégia empregada, foi possível selecionar e utilizar vários peptídeos representativos das enzimas da via glicolítica e também de algumas proteínas relevantes ao câncer. A utilização de peptídeos sintéticos facilitou substancialmente o processo de desenvolvimento do método. Por fim, com a metodologia desenvolvida, foi demonstrado para células MCF7, que o fator EGF alterou a expressão das proteínas da via glicolítica, aumento no fluxo para via das pentoses e capacidade aumentada da respiração celular. Este estudo, portanto, sugere uma nova disposição do metabolismo celular dado o conhecimento estabelecido em relação aos efeitos na respiração, como o efeito Warburg. / Cellular metabolism is altered during ontogenesis, cancer progression and several other pathological events. Because of that, the metabolism is constantly analyzed in order to provide further comprehension of processes that allow tissue growth, reproduction, homeostasis maintenance, and response to extracellular signals. Among the methods great diversity of methods used to characterize metabolic alterations, mass spectrometry has been contributing significantly to identify and quantify proteins involved in a diversity of metabolic pathways, but also to monitor the changes in metabolome. Proteomics based on mass spectrometry allows highthroughput in-depth qualitative resources for discovery phases stages, providing the new relevant candidates for further biochemical characterization. In a complementary way, targeted proteomics allows precise quantitative analyses of such selected protein targets. Here, it was developed a targeted proteomics method for multiplex monitoring glycolytic pathway enzymes and relevant cancer-related proteins. For that, proteotypic peptides representing proteins of interest were selected and studied in detail to be incorporated in a multiple reaction monitoring assay. The developed method was applied to monitor the alterations in the glycolytic pathway in a cancer progression model. Using targeted proteomics strategies, we selected and applied for quantification several proteotypic peptides representing glycolitic enzymes and cancerrelated proteins. The use of synthetic peptides allowed faster method development and more sensitive methods. The application of such methods, demonstrated alterations in glycolytic pathways and cancer-related proteins promoted in MCF7 cells treated with EGF. Also, an activation of pentose-phosphate pathway was suggested and increase in cellular oxygen consumption. This study, therefore, suggests changes in the cellular metabolism that differs from the classic Warburg effect observed during cancer development.

O metabolismo celular é substancialmente alterado durante a oncogênese, progressão tumoral e outros processos patológicos. Tem sido frequentemente analisado para compreensão dos processos que permitem o crescimento dos tecidos, reprodução, manutenção da homeostase e resposta a sinais extracelulares. Dentre os vários métodos para caracterização de alterações metabólicas, a espectrometria de massas tem contribuído significativamente para a identificação e quantificação de proteínas envolvidas no metabolismo, e também para a caracterização do metaboloma. A análise proteômica baseada em espectrometria de massas permite estudos qualitativos em grande escala, adequados para a busca e descoberta de analitos relevantes. A análise proteômica dirigida complementa esse caráter com qualidade quantitativa para proteínas alvo pré-selecionadas. Neste trabalho foram desenvolvidos métodos de proteômica dirigida para o monitoramento de alterações quantitativas nos níveis de proteínas envolvidas na via glicolítica do metabolismo da glicólise. Para tal peptídeos proteotípicos para cada proteína foram identificados e padronizados utilizando a estratégia de monitoramento de reações múltiplas. O painel foi aplicado para obter resultados das alterações que ocorrem em um modelo de progressão tumoral. Com esta estratégia empregada, foi possível selecionar e utilizar vários peptídeos representativos das enzimas da via glicolítica e também de algumas proteínas relevantes ao câncer. A utilização de peptídeos sintéticos facilitou substancialmente o processo de desenvolvimento do método. Por fim, com a metodologia desenvolvida, foi demonstrado para células MCF7, que o fator EGF alterou a expressão das proteínas da via glicolítica, aumento no fluxo para via das pentoses e capacidade aumentada da respiração celular. Este estudo, portanto, sugere uma nova disposição do metabolismo celular dado o conhecimento estabelecido em relação aos efeitos na respiração, como o efeito Warburg. / Cellular metabolism is altered during ontogenesis, cancer progression and several other pathological events. Because of that, the metabolism is constantly analyzed in order to provide further comprehension of processes that allow tissue growth, reproduction, homeostasis maintenance, and response to extracellular signals. Among the methods great diversity of methods used to characterize metabolic alterations, mass spectrometry has been contributing significantly to identify and quantify proteins involved in a diversity of metabolic pathways, but also to monitor the changes in metabolome. Proteomics based on mass spectrometry allows highthroughput in-depth qualitative resources for discovery phases stages, providing the new relevant candidates for further biochemical characterization. In a complementary way, targeted proteomics allows precise quantitative analyses of such selected protein targets. Here, it was developed a targeted proteomics method for multiplex monitoring glycolytic pathway enzymes and relevant cancer-related proteins. For that, proteotypic peptides representing proteins of interest were selected and studied in detail to be incorporated in a multiple reaction monitoring assay. The developed method was applied to monitor the alterations in the glycolytic pathway in a cancer progression model. Using targeted proteomics strategies, we selected and applied for quantification several proteotypic peptides representing glycolitic enzymes and cancerrelated proteins. The use of synthetic peptides allowed faster method development and more sensitive methods. The application of such methods, demonstrated alterations in glycolytic pathways and cancer-related proteins promoted in MCF7 cells treated with EGF. Also, an activation of pentose-phosphate pathway was suggested and increase in cellular oxygen consumption. This study, therefore, suggests changes in the cellular metabolism that differs from the classic Warburg effect observed during cancer development.

The goal of this research is to build an experimental environment for the Simulation Interoperability Laboratory (SIL) of the University of Central Florida (UCF). The Simulation Interoperability Laboratory (SIL) is researching about multi-resolution modeling(MRM), with a focus on military field uses. This thesis proposes steps to develop an MRM federation system and build two different MRM systems using COTS simulations (SIMBox, VR-Forces, and MASA Sword). This report is written to provide the basis for a time-based MRM federation study in the Simulation Interoperability Laboratory. The report describes many definitions and notions related to Multi-Resolution Modeling(MRM) and discusses examples to make better understanding for further research. MRM is relatively new research, and there are high demands for integrating simulators running in military field purposes. Most military-related research is based on simulators currently being used in the military; this poses a problem for research because the data is classified, resulting in many limitations for outside researchers to see the military's process for building an MRM system or the results of the research. Therefore, development of the MRM federation using COTS simulations can provide many examples of MRM issues for future research.

MALDI imaging allows the creation of a molecular image of a tissue slice. This image is reconstructed from the ion abundances in spectra that are obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method that can provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. My hypothesis was that a method based on multiple reaction monitoring (MRM) with isotopically-labelled internal standards can be developed which would allow the accurate quantitation of proteins in MALDI Imaging. This study reports on the development of this novel MALDI Imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in-situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically-labelled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI-MRM-MS and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabelled proteolytic peptides to the corresponding transitions from the applied isotopically-labelled standard peptides. In a parallel experiment, the quantity of the labelled peptide applied to the tissue was determined using a standard curve generated from MALDI-TOF-MS data. This external calibration curve was then used to extrapolate the quantity of endogenous peptide in a given area. All standard curves generated by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based imaging show the feasibility of obtaining precise and accurate quantitation of tissue protein concentrations over two orders of magnitude, while maintaining the spatial localization information for the proteins. / Graduate

Les oestrogènes font partie de la famille des neurostéroïdes. Notamment, l’Estradiol (17β-E2) pourrait avoirdes effets bénéfiques sur certaines anomalies liées à l'âge (déclin mnésique et cognitif, maladie d'Alzheimer). Le dosagede très faibles teneurs du 17β-E2 et de ces métabolites (Estrone, Estriol), dans les tissus cérébraux et le plasma, constitueun outil indispensable pour estimer la variation de leurs niveaux avec l'âge et dans différentes pathologies.Tout d'abord, nous avons élaboré une méthode en RP-HPLC-ESI(-)-MS/MS de quantification des oestrogènes sansdérivation chimique. La sélectivité de la séparation et la sensibilité de la détection de ces molécules ont été amélioréesaprès optimisation des paramètres MRM et chromatographiques.Puis, différents agents de dérivation ont été synthétisés et évalués afin d’augmenter le taux d’ionisation pour améliorerla sensibilité de détection. Après l’analyse ESI-MS et MS/MS de onze dérivés du 17β-E2, nous nous sommes intéressésplus particulièrement au dérivé 17β-E2-Q8S.Afin de réduire les interférences, une méthode de préparation de l’échantillon biologique (tissus cérébral, plasma desouris) a été développée. Les deux dosages (sans et avec dérivation) ont été comparées en termes de sensibilité, limitesde détection et de quantification (LD et LQ), linéarité et reproductibilité. Puis, elles ont été appliquées à l’analyse deséchantillons de cerveau, d'hippocampe et de plasma prélevés sur des animaux jeunes et âgés traités avec des quantitésconnues de 17β-E2. La méthode qui nous est apparue la plus robuste est la méthode sans dérivation avec une LD de0,5 fmole.μL-1. / Estrogens are neurosteroids, especially Estradiol (17β-E2) which is considered to be the most biologicallyactive form. 17β-E2 could have positive effects on some age-associated anomalies (memory and cognitive impairment,Alzheimer's disease). For elucidating and better understanding the molecular and cellular mechanisms that underliethe effects within the brain, it is necessary to quantitate 17β-E2 and its metabolites (estrone and estriol) in brain andplasma.First, the RP-HPLC-ESI(-)-MS/MS method without derivatization was developed. The selectivity of the separationand the sensitivity of detection of the estrogens has been improved after optimization of MRM and chromatographicparameters.Secondly, the various derivatization agents were evaluated after their synthesis in order to improve the sensitivity,selectivity and signal enhancement. After studying the eleven synthesized derivatives of 17β-E2 in ESI-MS andMS/MS, promising results were obtained with the 17β-E2-Q8S derivative.A simple purification method using liquid-liquid extraction followed by C18 solid phase extraction has been optimizedin order to minimize assay interferences. The two assays (with and without derivatization) were then compared interms of efficiency, detection and quantification limits (LOD/LOQ), calibration linearity and reproducibility. Then,both methods were validated on biological samples (brain, hippocampus and plasma) collected from animals treatedwith known amounts of 17β-E2. Finally, the more robust method was the method without derivatization with a LODof 0.5 fmol.μL-1.

Le nombre croissant de médicaments protéiques utilisés en thérapeutique a créé des besoins dans le domaine de leur quantification, principalement dans le plasma, un milieu de composition protéique complexe. Le dosage, essentiel aux études pharmacocinétique/pharmacodynamique, ainsi qu’à l’optimisation de ces traitements, est compliqué par la nature protéique de ces médicaments et par les faibles concentrations auxquelles ils sont attendus dans ces milieux complexes. La méthodologie proposée se démarque des méthodes de dosage usuelles par son caractère universel. Elle fait appel à la spectrométrie de masse adaptée à la quantification des protéines grâce à l’utilisation d’un marquage isotopique différentiel des peptides : après enrichissement et protéolyse, l’échantillon à doser est marqué sur les lysines par la version légère d’un réactif de dérivation. En parallèle, les peptides de la protéine médicament pure marqués par la version lourde du réactif, servent d’étalon interne. La possibilité de quantifier la protéine à partir de plusieurs de ses peptides améliore la fiabilité du dosage. Appliquée à l’epoetin beta aux concentrations attendues en thérapeutique (autour de 0,5 femtomole/µL de plasma), la stratégie proposée permet de situer la limite de quantification à environ 50 attomoles d’epoetin beta/µL de plasma avec une méthodologie de spectrométrie de masse nano-LC-ESI-Q-TRAP fonctionnant en mode MRM. Pour étendre l’universalité de cette approche au champ des protéines médicaments pégylées, une seconde molécule a été étudiée. Il s’agit de l’interféron alfa-2b pégylé qui a permis de mettre en place une stratégie d’extraction spécifique du médicament utilisant sa pégylation. / The growing number of therapeutic proteins has created needs in the field of their quantification, mainly in plasma, which is a complex protein environment. Quantitative analysis of these proteins is essential for pharmacokinetics/pharmacodynamics studies, and for the optimization of treatments. However, the nature itself of the analyte and the low concentrations that are expected in plasma complicate the quantitative analysis. The proposed methodology differs from usual methods on its universal applicability. It relies on mass spectrometry adapted to the quantification of proteins by using peptides differential isotope labelling : after enrichment and proteolysis, the therapeutic protein and the plasmatic proteins are labelled on lysine residues by the light reagent. In parallel, peptides of the pure therapeutic protein, labelled by heavy version of reagent, are used as internal standard. The ability to quantify the protein with several of its peptides improves the reliability of the analysis. When applied to epoetin beta at expected therapeutic concentrations (about 0.5 femtomole/µL of plasma), the proposed strategy leads to a quantification limit close to 50 attomoles of epoetin beta/µL plasma, with a nano-LC-ESI-Q-TRAP mass spectrometry methodology operating in MRM. To extend the universal character of this approach to the field of pegylated protein drugs, a second therapeutic protein model has been studied. This model is a pegylated interferon alfa-2b which allowed developing a strategy for specific extraction of the drug relying on its pegylation.

La quantification des anticorps monoclonaux (mAbs) dans le plasma est un pré-requis essentiel pour les études PK/PD. Les méthodes de références pour quantifier actuellement les mAbs sont de type ELISA mais les difficultés rencontrées notamment lorsque l’analyse porte sur des mAbs dont la cible pharmacologique est circulante, suggèrent que la spectrométrie de masse serait une alternative intéressante. Appliquée au bevacizumab, la stratégie développée fait appel à la spectrométrie de masse en tandem utilisée en mode MRM (HPLC-ESI-QqQ) et porte sur l’analyse des peptides spécifiques du bevacizumab obtenus à l’issu d’une protéolyse trypsique. La quantification absolue est réalisée à l’aide d’une droite de calibration obtenue à partir du ratio des aires des peptides du bevacizumab et de l’étalon interne. Afin de proposer une méthodologie de quantification de référence, nous avons définie les points clés du développement pour la transposition à d’autre mAbs et comparé les deux stratégies d’étalonnage interne les plus employées : l’une utilisant une protéine analogue et l’autre un peptide marqué par des isotopes stables (SIL-peptide). A travers ce développement la stratégie proposée présente un caractère universel vis-à-vis des anticorps monoclonaux de type IgG dont le traitement des échantillons repose sur une purification par protéine A suivit d’une concentration par ultrafiltration et dont la quantification fait appel à l’approche d’étalonnage interne SIL-peptide. Validée selon les recommandations de la FDA, notre méthode présente les performances analytiques attendues en termes de sensibilité, répétabilité et spécificité pour être appliquée à des études cliniques. / The quantification in plasma of monoclonal antibodies (mAbs) is an essential prerequisite to any PK/PD preclinical and clinical study. To date, reference techniques used to quantify mAbs, rely on enzyme-linked immunosorbent assay (ELISA) but the difficulties encountered in particular when the analysis focuses on the mAbs whose pharmacological target is circulating, suggest that mass spectrometry would be an interesting alternative. Applied to bevacizumab, the quantification developed strategy involves tandem mass spectrometry (HPLC-ESI-QqQ) used in MRM mode and focuses on the analysis of specific peptides bevacizumab obtained after tryptic proteolysis. Absolute quantification is achieved through calibration curve obtained from peak area ratios of bevacizumab surrogate peptide and internal standard. To propose a reference quantification methodology, we have identified the key points of development for transposition to other mAbs and compared the two most commonly used internal calibration approaches: one using protein analogue and the other a stable isotope labeled surrogate peptide (SIL-peptide). Through this development, the proposed strategy has a universal character with respect to IgG monoclonal antibodies subclasses which is based on sample processing purification using protein A followed by concentration by ultra filtration and whose quantification involves the internal calibration approach SIL-peptide. Validated according to FDA guidelines, our method shows the expected analytical performance in terms of sensitivity, specificity and repeatability for application in clinical studies

No description available.

Altered signaling pathways, which are mediated by post-translational modifications and changes in protein expression levels, are key regulators of cancer initiation, progression, and therapeutic escape. Many aspects of cancer progression, including early carcinogenesis and immediate response to drug treatment, are beyond the scope of genetic profiling and non-invasive monitoring techniques. Global protein profiling of cancer cell line models, tumor tissues, and biofluids (e.g. serum or urine) using mass spectrometry-based proteomics produces novel biological insights, which support improved patient outcomes. Recent technological advances resulting in next-generation mass spectrometry instrumentation and improved bioinformatics workflows have led to unprecedented measurement reproducibility as well as increased depth and coverage of the human proteome. It is now possible to interrogate the cancer proteome with quantitative proteomics to identify prognostic cancer biomarkers, stratify patients for treatment, identify new therapeutic targets, and elucidate drug resistance mechanisms. There are, however, numerous challenges associated with protein measurements. Biological samples have a high level of complexity and wide dynamic range, which is even more pronounced in samples used for non-invasive disease monitoring, such as serum. Cancer biomarkers are generally found in low abundance compared to other serum proteins, particularly at early stages of disease where cancer detection would make the biggest impact on improving patient survival. Additionally, the large-scale datasets generally require bioinformatics expertise to produce useful biological insights. These difficulties converge to create obstacles for down-stream clinical translation. This dissertation research demonstrates how proteomics is applied to develop new resources and generate novel workflows to improve protein quantification in complex biosamples, which could improve translation of cancer research to benefit patient care. The studies described in this dissertation move from assessment of quantitative mass spectrometry platforms, to analytical assay development and validation, and ending with personalized biomarker development applied to patient samples. As an example, four different quantitative mass spectrometry acquisition platforms are explored and comparisons of their ability to quantify low abundance peptides in a complex background are explored. Lung cancers frequently have aberrant signaling resulting in increased kinase activity and targetable signaling hubs; kinase inhibitors have been successfully developed and implemented clinically. Therefore, changes in amounts of kinase peptides in the complex background of peptides from all ATP-utilizing enzymes in a lung cancer cell line model after kinase inhibitor treatment was selected as a model system. Traditional mass spectrometry platforms, data dependent acquisition and multiple reaction monitoring, are compared to the two newer methods, data independent acquisition and parallel reaction monitoring. Relative quantification is performed across the four methods and analytical performance as well as downstream applications, including drug target identification and elucidation of signaling changes. Liquid chromatography – multiple reaction monitoring (LC-MRM) was selected for development of multiplexed quantitative assays based on superior sensitivity and fast analysis times, allowing for larger peptide panels. Method comparison results also provide guidelines for quantitative proteomics platform selection for translational cancer researchers. Next, a multiplexed quantitative LC-MRM assay targeting a panel of 30 RAS signaling proteins was developed and described. Over 30% of all human cancers have a RAS mutation and these cancers are generally aggressive and limited treatment options, leading to poor patient prognosis. Many targeted inhibitors have successfully shut down RAS signaling, leading to tumor regression, however, acquired drug resistance is common. The multiplexed LC-MRM assays characterized and validated are a publically available resource for cancer researchers to interrogate the RAS signal transduction network. Feasibility has been demonstrated in cell line models in order to identify signaling changes that confer BRAF inhibitor resistance and biomarkers of sensitivity to treatment. This analytical LC-MRM panel could support meaningful development of new therapeutic options and identification of companion biomarkers, with the end goal of improving patient outcomes. Multiplexed LC-MRM assays developed for personalized disease biomarkers using an integrated multi-omics approach are described for Multiple Myeloma, an incurable malignancy with poor patient outcomes. This disease is characterized by clonal expansion of the plasma cells in the bone marrow, which secrete a monoclonal immunoglobulin, or M-protein. Clinical treatment decisions are based on multiple semi-quantitative assays that require manual evaluation. In the clinic, minimal residual disease quantification methods, including multi-parameter flow cytometry and immunohistochemistry, are applied to bone marrow aspirates, which is a highly invasive technique that does not provide a systemic evaluation of the disease. To address these issues, we hypothesized that unique variable region peptides could be identified and LC-MRM assays developed specific to each patient’s M-protein to improve specificity and sensitivity in non-invasive disease monitoring. A proteogenomics approach was used to design personalized assays for each patient to monitor their disease progression, which demonstrate improved specificity and up to a 500-fold increase in sensitivity compared to current clinical methods. Assays can be developed from marrow aspirates collected when the patient was at residual disease stage, which is useful if no sample with high disease burden is available. The patient-specific tests are also multiplexed with constant region peptide assays that monitor all immunoglobulin heavy and light chain classes, which could reduce analysis to a single test. In conclusion, highly sensitive patient-specific assays have been developed that could change the paradigm for patient evaluation and clinical decision-making, increasing the ability of clinicians to continue first line therapy in the hopes of achieving a cure, or to intervene at an earlier time point in disease recurrence. This study also provides a blueprint for future development of personalized diagnostics, which could be applied to biomarkers of other cancer types. Overall, these studies demonstrate how quantitative proteomics can be used to support translational cancer research, from the impact of different mass spectrometry platforms on elucidating signaling changes and drug targets to the characterization of multiplexed LC-MRM assays applied to cell line models for translational research purposes and in patient serum samples optimized for clinical translation. We believe that mass spectrometry-based proteomics is poised to play a pivotal role in personalized diagnostics to support implementation of precision medicine, an effort that will improve the quality and efficiency of patient care.

<p>Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum.</p><p>The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with <em>tert</em>-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible.</p>