Lipidomics is a relatively new field under the heading of systems biology. Due to its infancy, the field suffers from significant ‘growing pains’, one of which is the lack of bioinformatic analytic resources that other “-omics” fields enjoy. Here, I describe the creation and validation of the glycerophospholipid identification program VaLID. Using an in silico approach, we generated a comprehensive database containing all of the glycerophospholipids within multiple sub-classes: those containing chains of 0 to 30 carbons with up to 6 unsaturations and various linkages. Using Java, I created a web- based computer interface with a search engine and a visualization tool to access this database. In comparing results to current programs, I found that VaLID consistently contained more identity predictions than did the current gold standard LipidMAPS. Results from several tests with real datasets confirm that VaLID is more than capable as a phospholipid identification tool for use in lipidomics.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted glycoprotein that promotes degradation of low-density lipoprotein receptors. Gain- and loss-of-function variants of PCSK9 cause hypercholesterolemia and hypocholesterolemia, respectively. Although it has been a decade since the discovery of PCSK9, its effect in terms of global protein changes and interactions still require further understanding. This study provided a global outlook at the protein changes caused by PCSK9 and its variants in human hepatic HUH7 cell line. First, a proteomics-based method for protein subcellular distribution analysis has been developed. Second, through secretome analyses, six apolipoproteins and six proteins involved in the coagulation pathway were found with >2-fold changes between wild type PCSK9 and its variants. Third, through secreted interactome analyses, a list of 159 PCSK9 interactor candidates was identified. Two interacting proteins, FASN and PSMD2, were validated and demonstrated with dynamic interacting patterns between PCSK9 and its variants.

We have built an imaging mass spectrometer adapted for ultrashort laser pulses as its ionization technique, as an alternative to other imaging techniques. Before my arrival, the mass spectrometer has only been subject to preliminary tests on noble gases. Since then, we’ve made some modifications to the system in order to properly analyze solids. This thesis shows how we obtain our ultrashort laser pulses, the inner workings of our homemade imaging mass spectrometer, and the results that we’ve obtained with it so far. We tested two modes of operation concerning the extraction of the ions from the system into the mass analyzer: continuous and pulsed. We discuss the advantages and disadvantages of each configuration. We also display preliminary imaging results with our imaging technique of a simple WO3 and ITO structure. We conclude by comparing the resolution of this image to the different techniques in imaging mass spectrometry, how we can further improve our mass spectrometer, and the future use of this machine. Nous avons construit un spectromètre de masse adapté pour les pulses de laser très courts comme technique d’ionisation, pour acquisition des images d’un échantillon. Avant je suis arrivé, le spectromètre de masse avait seulement été utilisé pour des tests préliminaires de gaz nobles. Depuis ce moment, nous avons modifié le système pour analyser les solides. Cette thèse démontre comment on obtient nos pulses de laser très courts, comment notre spectromètre fait maison fonctionne et les résultats nous avons obtenus jusqu’à présent. Nous avons testé deux configurations différentes au sujet de l’extraction des ions du système : constant et pulsé. Nous discutons aussi les avantages et désavantages de chaque mode d’opération. Nous démontrons aussi des images préliminaires d’un substrat mixte de WO3 et ITO. Nous concluons par comparer la résolution des images aux autres techniques de collection d’images, comment nous pouvons améliorer notre spectromètre de masse et les plans pour la machine dans le futur.

In recent years, the role of metabolomics has become increasingly more important in the advancement of many research fields including medical studies. Due to lack of metabolomics research in the area of infectious disease and the rise in antibiotic resistance, there is a need for further studies on the modes of antibiotic action and the mechanisms of resistance of pathogenic microorganisms at the metabolome level. This study aimed to investigate effects of DNA synthesis inhibitors on the metabolome of E. coli and to develop a workflow for discrimination between E. coli isolates down to the sub-species level using a variety of methods, which can inform the choice of analytical techniques in metabolomics research. A metabolomics-based approach was used to elucidate metabolic alterations in E. coli K-12 upon challenge with trimethoprim at two pH levels (5 and 7) which mimic human urine acidity. FT-IR spectroscopy was used as a preliminary experiment to produce bacterial fingerprints and GC-MS was applied to generate global metabolic profiles in each condition. At pH 7, as the drug molecules exhibited higher permeability, stronger direct effects of the antibiotic were observed, i.e. decreased levels of nucleotides. Trehalose, an osmoprotectant, was up-regulated in these stress conditions and this up-regulation was mirrored by a decrease in glucose levels. This also correlated with up-regulation of pyruvate-related products (e.g. alanine, citrate and malate). Other off-target related effects were observed such as alterations in the levels of various amino acids upon trimethoprim challenge. This study offered a wider view of drug action at pH levels similar to healthy human urine. A high throughput FT-IR spectroscopy method was developed to discriminate between pathogenic E. coli isolates based on sequence type. This method employed a Bioscreen as a micro-culture incubator instead of traditional sample preparation (shaking flasks), which can be labour intensive and time consuming. Excluding the washing step in the protocol enabled discrimination between isolates of different sequence types. Moreover, a reproducible workflow of lipid analysis based on LC-MS was developed and applied on four pathogenic isolates with different sequence type and susceptibility to ciprofloxacin. This workflow enabled detection of a wide range of lipid classes and determination of significant alterations in lipid levels related to susceptibility to ciprofloxacin. Stressed and control isolates were also analysed using the developed Bioscreen FT-IR approach to assess phenotypic fingerprint differences, which were in line with the LC-MS-ve class distribution. Further investigation by means of four analytical platforms (FT-IR, GC-MS, LC-MS-ve and LC-MS+ve) was applied on E. coli ST131 isolates characterised using classical microbiological tests (virulence factors and metabolic tests). Procrustes transformation was used to compare between the analytical methods and the microbiological tests in terms of their capacity to discriminate between the different isolates. As indicated above, the results from FT-IR and LC-MS-ve were comparable and in line with virulence tests, while GC-MS and metabolic tests were in agreement. Complementary information generated by different analytical techniques and microbiological tests may indicate the requirement for careful selection of the method of investigation and may suggest the need to continue using a combination of methods which are applied to study different features of bacterial physiology.

The field of proteogenomics operates at the interface between proteomics and genomics, and has emerged during the past decade to exploit the vast quantities of high-throughput sequence data. A range of different proteogenomics approaches have been developed, which integrate mass spectrometry data with genome sequence data to provide empirical evidence for protein-coding genes. However, current methods may not be optimized as they do not fully consider the splicing complexity in eukaryotes and there is currently no best practice method. To address this, we investigate the level of proteomics support for Ensembl gene models in human, and a selection of model organisms. We find a disparity between the number of splice variants confirmed by extant data, and the number that can theoretically be confirmed using current proteomics technologies. We then go on to investigate EST-based proteogenomics methods, which enabled the discovery of novel peptide sequences in the chicken genome, which represent hitherto unannotated genes, amended gene models, polymorphisms, and genes missing from the genome assembly. Different approaches for searching mass spectrometry data against transcript sequences are explored, and we show that searching mass spectra against protein sequences predicted by the EORF and ESTScan2 translation tools results in the best sensitivity.

Cell adhesion to the extracellular matrix is mediated by the integrin family of adhesion receptors. Integrin receptor engagement initiates the formation of multimolecular protein complexes, termed integrin adhesion complexes (IACs), at the cell membrane. IACs are complex signalling hubs that are enriched in tyrosine-based phosphorylation events and form a mechanochemical connection between integrin receptors and the actin cytoskeleton. Dysregulation of individual IAC components has been reported to influence a wide range of biological processes that contribute to disease. Literature-curated and proteomic analyses of IACs have revealed an unanticipated molecular complexity of IACs in a variety of experimental contexts; however, a global consensus view of the composition of IACs, and a description of how the complex network of interactions in IACs influences global cell function, is currently lacking. Here, multiple existing and new proteomic datasets detailing the protein composition of IACs were analysed to identify a systems-level description of IACs and to enable interrogation of IAC structure, topology and dynamics. Quantitative IAC proteomes derived from multiple cell types were integrated to generate a 2,412-protein ‘meta-adhesome’ database of proteins enriched to fibronectin-induced IACs. To investigate the putative functional adhesion landscape in an objective manner, the meta-adhesome was analysed using a combination of hierarchical clustering, gene ontology and interaction network analyses. An emergent property of the meta-adhesome was the definition of a consensus adhesome: 60 proteins commonly identified from IAC datasets that likely represent an IAC protein core composition. The consensus adhesome highlights how integrins connect to actin via multiple pathways and consists of both canonical and underappreciated IAC components. To investigate the robustness of the IAC network, the effects of pharmacological perturbation of the key IAC kinases FAK and Src on IACs were examined. FAK activity was inhibited with the small molecule inhibitor AZ13256675, and mass spectrometry-based protein quantification revealed that IAC protein composition was unaffected upon FAK inhibition. Moreover, IAC composition was also insensitive to Src inhibition using AZD0530 and to simultaneous FAK and Src inhibition. In contrast, phosphorylation of IAC components, cell migration and cell proliferation were reduced upon FAK and/or Src inhibition. These data suggest that IAC protein composition is robust to perturbation of key kinases, while flux of signals propagated through IACs via phosphorylation is kinase dependent. To examine IAC dynamics, the composition of IACs during IAC assembly and IAC disassembly were examined in the context of the meta-adhesome and consensus adhesome using IAC proteomic datasets. These analyses revealed the temporal dynamics of specific functional protein modules at IACs and detailed the compositional dynamics of the core cell adhesion machinery. In summary, these studies describe both a systems-level and a reductionist view of the IAC proteome, investigate the effects of kinase inhibition on IAC composition and chart IAC dynamics during their assembly and disassembly. These data demonstrate the usefulness of the meta-adhesome and consensus adhesome for future analyses of IAC proteomes.

Polymeric nanoparticles were synthesized and loaded with Cu²⁺ to explore the therapeutic potential for catically active transition metal ions and complexes other than cisplatin. Two types of nanoparticles were synthesized to show the potential for polymer based vectors. Copper loading and release were characterized via inductively coupled plasma mass spectrometry (ICP MS), energy dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), and elemental analysis. Results demonstrated that Cu could be loaded to the nano-sized carriers in an aqueous environment, and that the release was pH-dependent. The toxicity of these particles was measured in HeLa cells where significant toxicity was observed in vitro via dosing of high Cu-loaded nanoparticles. No significant toxicity was observed in cells dosed with Cu-free nanoparticles.

Orientador: Fábio Cesar Gozzo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T16:09:31Z (GMT). No. of bitstreams: 1 Koolen_HectorHenriqueFerreira_D.pdf: 16550075 bytes, checksum: 0fafb8ced0640f9080c108d7cb303ac4 (MD5) Previous issue date: 2015 / Resumo: A espectrometria de massas (MS) consiste no estudo de íons na fase gasosa. O grande salto na utilização da MS devido ao desenvolvimento dos métodos de ionização por eletrospray (ESI), por J. B. Fenn e ionização/dessorção a laser auxiliada por matriz (MALDI) por M. Karas e F. Hillenkamp no final da década de 80 abriu espaço para o desenvolvimento de novos equipamentos, desta forma expandindo em muito a capacidade de análise de diversos analitos. Misturas complexas constituem um desafio até hoje, onde diversas técnicas são empregadas com o objetivo de se entender a constituição destas matrizes. Neste contexto a MS vêm sendo utilizada em conjunto com a mobilidade iônica (IM) em estudos com diversos analitos, como por exemplo, em proteômica e petroleômica. Este trabalho é constituído por dois capítulos, onde na primeira parte as técnicas foram utilizadas em experimentos de proteômica estrutural por meio de ligação cruzada. Estudos Fundamentais foram conduzidos de modo a serem exploradas as misturas complexas de peptídeos provenientes de experimentos utilizando-se uma classe pouco explorada de reagentes de ligação cruzada (imidoésteres). Para o segundo capítulo a mesmas técnicas foram utilizadas nas análises da fração pesada (asfaltenos) de petróleos brasileiros. A simplificação das amostras juntamente com a caracterização estrutural destes compostos por meio de suas sessões de choque de colisão (CCS) constituiu a estratégia de análise para este capítulo. Como resultados da primeira parte têm-se o estabelecimento das rotas de fragmentação de peptídeos modificados pelo reagente de ligação cruzada dimetil suberoimidato e a aplicação do mesmo em experimentos com sistemas proteicos reais. A IM foi aplicada de modo a se separar as espécies geradas após os experimentos reacionais dos peptídeos não modificados por meio de seus estados de carga o que possibilitou, uma rápida e simplificação deste tipo de mistura. Como resultados da segunda parte desta tese a IM foi empregada na caracterização das CCS de compostos modelo de asfaltenos e posteriormente em íons representativos da fração pesada. Os resultados foram validados mediante cálculos teóricos e espectrometria de massas de ultra-alta resolução. Os dados obtidos vão de encontro com os modelos de estruturas de arquitetura do tipo ilha previstos no modelo de Yen-Mullins, constituindo desta forma o primeiro estudo estrutural de asfaltenos usando-se estas técnicas combinadas / Abstract: Mass spectrometry (MS) constitutes the study of ions in the gas-phase, being the structural elucidation the main application of MS. With the development of ionization techniques such as electrospray (ESI), by J. B. Fenn and matrix assisted laser desorption by M. Karas and F. Hillenkamp in the later 80¿s allowed the development of new instruments, providing an expansion of the array of analytes suitable for MS analysis. Complex mixtures constitute a challenge nowadays, where diverse techniques are employed with the objective of understanding the chemical composition of such matrixes. In this sense MS have been used along with ion mobility (IM) in the study of different analytes (e.g. in Proteomics and Petroleomics). This works comprises two chapters, where in the first one the techniques were applied in structural proteomics by means of chemical cross-linking experiments. Fundamental studies were carried to explore complex mixtures of tryptic peptides after experiments with imidoesters reagents. For the second chapter the same techniques were applied in the analysis of the polar fraction of Brazilian petroleum¿s (asphaltenes), where beyond separations an structural study was carried out to verify the existing models regarding asphaltene structure. As results of the chapter 1 the behavior at the gas-phase of the peptides upon dissociation techniques was elucidated (fragmentation studies). The validation of imidates as cross-linking reagents was performed to real proteins. IM was applied to separate different charge states created by the modified peptides generated by the imidate reagent. As results from the second chapter IM was successfully applied, were the comparison with model compounds was done employing also other MS techniques such as ultra-high resolution (UHR) MS. The findings of this chapter reinforce the Yen-Mullins model that describes asphaltenes as compounds possessing island structures / Doutorado / Quimica Organica / Doutor em Ciências

>Magister Scientiae - MSc / Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of a single highly connected interactor can cause widespread disruption of cellular processors including diseases and cancer. Therefore, detailed study of the interactions made by cancer-related proteins will help explain their role in the interaction networks. The investigation of proteins by mass spectrometry (MS) has provided unique opportunities to gain insight into the dynamics of these proteins at the molecular level. MS uses mass analysis for protein characterization, and is currently the most comprehensive and versatile tool in proteomics. MS can provide confirmation of protein samples of interest, accurate molecular mass measurements of proteins, purity of protein samples, detection of posttranslational modifications, and more recently, interactions between two or more proteins. The conventional way of investigating the structure of proteins involves nuclear magnetic resonance (NMR) or X-ray crystallography. Compared to MS these methods are time consuming methods and, furthermore, require a considerable amount of protein. MS has proved to be useful in this regard as it provides insights into the structural arrangement of proteins, and/or their interacting partners, without the need for crystalliastion or the tedious process of backbone assignment before structural and functional annotations can be attributed to the protein of interest. However, in many cases, conventional methods are used parallel to MS to serve as validation of the MS data. The broad objective of this MSc study was to provide structural and functional insights into the function of Retinoblastoma Binding Protein-6 (RBBP6), using a MS approach. The aims were twofold: 1) to investigate metal ion binding by RING (Really Interesting New Gene) finger domains from RBBP6, and 2) to investigate the in vitro interaction between RBBP6 and Hsp 70(Heat Shock Protein 70), and between RBBP6 and Murine Double Minute-2 (Mdm2).

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.