The common concept in this thesis was to adapt and develop quantitative mass spectrometric assays focusing on reagents originating from the Human Protein Atlas project to quantify proteins in human cell lines, tissues and blood. The work is based around stable isotope labeled protein fragment standards that each represent a small part of a human protein-coding gene. This thesis shows how they can be used in various formats to describe the protein landscape and be used to standardize mass spectrometry experiments. The first part of the thesis describes the use of antibodies in combination with heavy stable isotope labeled antigens to establish a semi-automated protocol for protein quantification of complex samples with fast analysis time  (Paper~I). Paper II introduces a semi-automated cloning protocol that can be used to selectively clone variants of recombinant proteins, and highlights the automation process that is necessary for large-scale proteomics endeavors. This paper also describes the technology that was used to clone all protein standards that are used in all of the included papers.                       The second part of the thesis includes papers that focus on the generation and application of antibody-free targeted mass spectrometry methods. Here, absolute protein copy numbers were determined across human cell lines and tissues (Paper III) and the protein data was correlated against transcriptomics data. Proteins were quantified to validate antibodies in a novel method that evaluates antibodies based on differential protein expression across multiple cell lines (Paper IV). Finally, a large-scale study was performed to generate targeted proteomics assays (Paper V) based on protein fragments. Here, assay coordinates were mapped for more than 10,000 human protein-coding genes and a subset of peptides was thereafter used to determine absolute protein levels of 49 proteins in human serum.                       In conclusion, this thesis describes the development of methods for protein quantification by targeted mass spectrometry and the use of recombinant protein fragment standards as the common denominator. / <p>QC 20161013</p>

A variety of analytical techniques are showcased for their ability to provide insights into reaction mechanisms as well as active intermediate speciation. Pressurized Sample Infusion-Mass Spectrometry (PSI-ESI-MS), ion mobility-mass spectrometry (IMS-MS), and Nuclear Magnetic Resonance (NMR) spectroscopy are powerful analytical techniques capable of reaction monitoring. Contamination from vulcanized rubber was an issue with the PSI-ESI-MS technique as ions unrelated to the reaction were convoluting the mass spectrum. This was resolved by re-designing the PSI flask such that the septum was positioned above a condenser, preventing heat degradation of the septum and subsequent leaching of contam- inants into the reaction solution. The technique was then used to analyze the Buchwald-Hartwig amination reaction in real-time. The innovative use of Multiple Reaction Monitoring (MRM) scans facilitated observation of all catalytic intermediates, and elucidation of relative reaction rates for each step of the catalytic cycle. PSI-ESI-MS and NMR are complementary methods whereby catalytic intermediates are monitored via PSI-ESI-MS, and the rate of product formation is monitored via NMR spectroscopy. This combination of analytical methods was employed in the investigation of the Barluenga cross-coupling reaction between N-tosylhydrazones and aryl halides. A reaction screen revealed optimized homogeneous conditions, and the turnover limiting step was found to be off-cycle. IMS separates gaseous ions based on their size and shape immediately prior to MS analysis. Upon investigation of [PtCl3(C2H4)], and [PtCl3(CO)], it was found that residual [PtCl3] was forming [PtCl3(N2)] in the source of the instrument. Ion mobility was able to separate these isobaric ions, and DFT calculations and collision-induced dissociation experiments confirmed the existence of the gaseous [PtCl3(N2)] complex. NMR spectroscopy may also be employed as a strong reaction monitoring technique. The mechanism of C–H silylation by trimethyl(trifluoromethyl)silane and tetrabutylammonium difluorotriphenylsilicate was investigated using 19F-NMR. All intermediates and reaction byproducts were quantitatively observed, and the reaction conditions were optimized. A stopped-flow NMR system was used to gather data points in the first 0.2 seconds of the reaction. / Graduate

No description available.

An optimized method for the chiral resolution of enantiomers of amino acids in bacterial supernatants is reported. This LC-MS/MS method is performed using a chiral Teichoplanin LC column and does not require sample clean up or chemical derivitization. This method allows for the determination of the relative amounts of the D and L enantiomers of 20 proteinogenic amino acids. The detection limits and response factors for the 20 amino acids were determined. Calibrations over three orders of magnitude showed least squares coefficient values (R^2) greater than 0.996 for eighty percent of the amino acids and greater than 0.992 for the remainder. The amino acids and their enantiomers were identified based on their retention times and their unique Multiple Reaction Monitoring (MRM) transitions for each amino acid. L-Aspartic acid-2,3,3-d3 was used as the internal standard. Cultures of Sinorhizobium meliloti (a nitrogen-fixing soil bacterium) were grown on minimal media; thus, all amino acids were biosynthesized by the bacterium. After centrifugation, supernatants were freeze dried, reconstituted in a small volume of methanol/water with internal standard and injected onto the LC column. The amino acids detected in the bacterial supernatant and the concentrations of the enantiomers were reported as the L and D isomers respectively: arginine [L, 12.6 ± 3.1 μg/L; D, 10.1 ± 3.2 μg/L], serine [L, 7.2 ± 1.16 μg/L; D, n.d.], threonine [L, n.d.; D, 11.2 ± 2.7 μg/L] and valine [L, 15.5 ± 4.3 μg/L; D, 11.3 ± 3.7 μg/L], where the term n.d. means below detection limit. The limits for detection for all amino acids ranged from 1.3 μg/L - 5.1 μg/L. In media with no added phosphate, the amino acid profiles changed somewhat under these stress conditions. Arginine was no longer detected while alanine and proline were now observed; the concentrations of the amino acids were: alanine [L, 7.7 ± 1.2 μg/L; D, 13.4 ± 2.5 μg/L], proline [L, n.d.; D, 8.63 ± 1.3 μg/L], serine [L, 7.6 ± 1.2 μg/L; D, n.d.], threonine [L, n.d.; D, 10.2 ± 3.2 μg/L] and valine [L, 11.6 ± 2.3 μg/L; D, 10.1 ± 3.1 μg/L]. These data represent the mean values of three independent bacterial growth experiments conducted over a 3 month period; the data came from the analysis of five separate aliquots from each growth experiment. The percent standard deviation for these data ranged from 15% to 33% and averaged 24%. Under both the normal and stressed growth conditions of S. meliloti produced the L enantiomer of serine, the D enantiomer of threonine and racemic valine. While racemic arginine was observed under normal growth conditions, levels were below detection under stressed conditions; under stress conditions only the D enantiomer of proline was observed while alanine was found in 1:2, L:D ratio. / Thesis / Master of Science (MSc)

A biotecnologia é definida como qualquer aplicação tecnológica que utilize sistemas biológicos, organismos vivos, ou seus derivados, para fabricar ou modificar produtos ou processos para alguma utilização específica (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Os produtos biotecnológicos devem atender certas especificações exigidas pela Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA) e Wood Health Organization (WHO). Para isso, utilizam-se técnicas rotineiras aplicadas à pesquisa e análise de biomoléculas como Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) e espectrometria de massas (MS). A abordagem do Monitoramento de Reações Múltiplas (MRM) é considerada uma interessante alternativa aos ensaios imunoenzimáticos para caracterização e quantificação total de proteínas terapêuticas, sejam elas recombinantes ou não (KIM e DOYLE, 2010). Esta abordagem é baseada nos fundamentos da proteômica quantitativa pela utilização da cromatografia líquida acoplada à espectrometria de massas sequencial (LCMS/MS) cuja plataforma apresenta alta especificidade, sensibilidade e reprodutibilidade quantitativa em suas análises para detecção simultânea de várias regiões da estrutura proteica. Em vista disso, estabeleceu-se uma metodologia para quantificação do FVIII recombinante (FVIIIr) produzido pela linhagem celular humana Sk-Hep-1 ou do FVIII derivado do plasma (FVIIIdp), ambos utilizados no tratamento da Hemofilia A (HEMA). Para o estabelecimento da metodologia, toda uma estratégia de seleção e síntese química de peptídeos representativos das cadeias pesada e leve do FVIII humano foi empregada. Tais peptídeos obtidos foram utilizados como padrões das análises. Além disso, foi demonstrada a relação direta entre a quantificação total do FVIII com técnicas convencionais como ELISA, possibilitando a aplicação rotineira dessa abordagem para quantificação do FVIIIr e do FVIIIdp. / Biotechnology is defined as any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for a specific use (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Biotechnological products must meet certain specifications required by Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA), and Wood Health Organization (WHO). For that routine techniques applied to research and analysis of biomolecules such Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA), and mass spectrometry (MS) are utilized as quality control techniques. The MS-based approach termed Multiple Reaction Monitoring (MRM) is considered an interesting alternative to immunoassays for the total quantification and characterization of therapeutic proteins, whether recombinant or not (KIM e DOYLE, 2010). This approach is based on the fundamentals of quantitative proteomics and uses of liquid chromatography coupled to tandem spectrometric mass (LC-MS/MS) to obtain a highly specific, sensitive and reproducible analysis for quantification of multiple regions of a given protein structure. Here, we established a methodology for total quantification of recombinant FVIII (rFVIII) produced in Sk-Hep-1 human cell line or of plasma derived FVIII (pdFVIII), both used in the treatment of Hemophilia A (HEMA). For that, we adopted a strategy of selection and chemical peptide synthesis of representative peptides of the heavy and light chains of human FVIII, which were used as standards in the analysis. The quantitative MRM method developed here indicated a direct correlation with FVIII quantitative techniques such ELISA, which allows routine application of such approach for rFVIII and pdFVIII quantification.

Orientador: Marcos Nogueira Eberlin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-18T08:57:37Z (GMT). No. of bitstreams: 1 Santos_VanessaGoncalvesdos_M.pdf: 2677797 bytes, checksum: df960f3d5c05d314477c8333f88eb71a (MD5) Previous issue date: 2011 / Resumo: Um estudo de mecanismo de reação, seja ele por espectrometria de massas ou por outras técnicas, deve garantir que as propostas estejam o mais próximo possível do mecanismo da reação estudada. Para isso o estudo deve reproduzir as condições experimentais para esta reação com a mínima interferência, deste modo, o uso de aditivos (sejam eles ácidos ou básicos) para protonar ou desprotonar os intermediários de reação podem perturbar o caminho reacional ou mesmo o equilíbrio da reação em estudo. Além disso, o favorecimento de uma espécie em relação as demais, de acordo com a afinidade por prótons, pode dificultar a detecção de alguns dos intermediários de interesse, prejudicando assim a interpretação do mecanismo. Sendo assim, o presente trabalho tem como objetivo propor novas metodologias para o estudo de mecanismos de reações orgânicas por espectrometria de massas, de forma buscar soluções e contornar problemas inerentes da técnica. No presente trabalho, foi avaliado a utilização de reagentes carregados, (não ácidos e não básicos) não sendo necessário, neste caso, o uso de aditivos, uma vez que todos os intermediários irão carregam uma ¿etiqueta¿ de carga que facilita sua detecção por espectrometria de massas. Estes reagentes marcados foram utilizados no estudo do mecanismo da reação multicomponente de Hantzsch. Além disso, uma nova fonte de ionização ambiente foi desenvolvida com o propósito de facilitar estudos online de mecanismos reacionais, a qual se mostrou útil sendo útil não apenas para monitorar o andamento da reação, mas também para um estudo mais detalhado do mecanismo, permitindo a detecção de intermediários transientes presentes no meio reacional que são de extrema importância para a elucidação do mecanismo da reação. Por fim, a nova fonte foi empregada no estudo online da reação multicomponente de Hantzsch de forma a avaliar cineticamente o mecanismo anteriormente estudado por ESI-MS(/MS) / Abstract: A study of reaction mechanisms, either by mass spectrometry or by another technique, should ensure that proposals are similar the possible mechanism of the reaction studied. For this study should accurately reproduce the experimental conditions for this reaction with minimal interference. Thereby, using additives (acids or basics) to protonated or deprotonated the intermediates in solution can disturb the reaction path or even the equilibrium of the reaction studied. Moreover, the encouragement of a species in relation to others, according to the affinity for protons, may hinder the detection of some of the intermediate of interest, thus hampering interpretation of the mechanism. This dissertation aims to propose new methodologies for studying the mechanisms of organic reactions by mass spectrometry, in order to seek solutions and to get over problems inherent in the technique. Thus, a new ionization source was developed aiming to became possible online mechanisms studies, this new source could be useful not only to monitor the progress of the reaction but also for a more detailed study of the mechanism, allowing the transient intermediates detection on solution, which are important to mechanism elucidation. Moreover, this source was applied in the analysis of commercial samples like drugs, alcoholics drinks and crude oil, in order to show its efficacy either to pure samples and complex matrices. Our work proposes also the use of charged tags in order to perform analysis without additives (acids or basics), since all intermediates are charged, making it easy its detection by mass spectrometry. These charged tags are going to be used in the investigation concerning the multicomponent Hantzch reaction / Mestrado / Quimica Organica / Mestre em Química

A biotecnologia é definida como qualquer aplicação tecnológica que utilize sistemas biológicos, organismos vivos, ou seus derivados, para fabricar ou modificar produtos ou processos para alguma utilização específica (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Os produtos biotecnológicos devem atender certas especificações exigidas pela Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA) e Wood Health Organization (WHO). Para isso, utilizam-se técnicas rotineiras aplicadas à pesquisa e análise de biomoléculas como Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA) e espectrometria de massas (MS). A abordagem do Monitoramento de Reações Múltiplas (MRM) é considerada uma interessante alternativa aos ensaios imunoenzimáticos para caracterização e quantificação total de proteínas terapêuticas, sejam elas recombinantes ou não (KIM e DOYLE, 2010). Esta abordagem é baseada nos fundamentos da proteômica quantitativa pela utilização da cromatografia líquida acoplada à espectrometria de massas sequencial (LCMS/MS) cuja plataforma apresenta alta especificidade, sensibilidade e reprodutibilidade quantitativa em suas análises para detecção simultânea de várias regiões da estrutura proteica. Em vista disso, estabeleceu-se uma metodologia para quantificação do FVIII recombinante (FVIIIr) produzido pela linhagem celular humana Sk-Hep-1 ou do FVIII derivado do plasma (FVIIIdp), ambos utilizados no tratamento da Hemofilia A (HEMA). Para o estabelecimento da metodologia, toda uma estratégia de seleção e síntese química de peptídeos representativos das cadeias pesada e leve do FVIII humano foi empregada. Tais peptídeos obtidos foram utilizados como padrões das análises. Além disso, foi demonstrada a relação direta entre a quantificação total do FVIII com técnicas convencionais como ELISA, possibilitando a aplicação rotineira dessa abordagem para quantificação do FVIIIr e do FVIIIdp. / Biotechnology is defined as any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for a specific use (ORGANIZAÇÃO DAS NAÇÕES UNIDAS, 1992). Biotechnological products must meet certain specifications required by Agência Nacional de Vigilância Sanitária (ANVISA), Food and Drug Administration (FDA), European Medicine Agency (EMEA), and Wood Health Organization (WHO). For that routine techniques applied to research and analysis of biomolecules such Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Western blot, Enzyme-Linked Immunosorbent Assay (ELISA), and mass spectrometry (MS) are utilized as quality control techniques. The MS-based approach termed Multiple Reaction Monitoring (MRM) is considered an interesting alternative to immunoassays for the total quantification and characterization of therapeutic proteins, whether recombinant or not (KIM e DOYLE, 2010). This approach is based on the fundamentals of quantitative proteomics and uses of liquid chromatography coupled to tandem spectrometric mass (LC-MS/MS) to obtain a highly specific, sensitive and reproducible analysis for quantification of multiple regions of a given protein structure. Here, we established a methodology for total quantification of recombinant FVIII (rFVIII) produced in Sk-Hep-1 human cell line or of plasma derived FVIII (pdFVIII), both used in the treatment of Hemophilia A (HEMA). For that, we adopted a strategy of selection and chemical peptide synthesis of representative peptides of the heavy and light chains of human FVIII, which were used as standards in the analysis. The quantitative MRM method developed here indicated a direct correlation with FVIII quantitative techniques such ELISA, which allows routine application of such approach for rFVIII and pdFVIII quantification.

(1) Introduction: Sulfonates, which can be diet- or host-derived, are a class of compounds detected in the gut, are involved in host–microbiome interactions and have several health effects. Our aim was to develop a method to quantify five of the sulfonates in the intestine and apply it in a simplified human microbiome model. These were taurine, its metabolic precursor cysteate and one of its degradation products isethionate, as well as sulfoquinovose and one of its most relevant degradation products 2,3-dihydroxy-1-propanesulfonate. (2) Methods: An extraction and sample preparation method was developed, without the need for derivatization. To detect and quantify the extracted sulfonates, a multiplexed LC-MS/MS-MRM method was established. (3) Results: The accuracy and precision of the method were within GLP-accepted parameters. To apply this method in a pilot study, we spiked either taurine or sulfoquinovose into an in vitro simplified human microbiota model with and without Bilophila wadsworthia, a known sulfonate utilizer. The results revealed that only the culture with B. wadsworthia was able to degrade taurine, with isethionate as an intermediate. After spiking the communities with sulfoquinovose, the results revealed that the simplified human microbiome model was able to degrade sulfoquinovose to 2,3-dihydroxypropane-1-sulfonate, which was probably catalyzed by Escherichia coli. In the community with B. wadsworthia, the 2,3-dihydroxypropane-1-sulfonate produced was further degraded by B. wadsworthia to sulfide. (4) Conclusions: We successfully developed a method for sulfonate quantification and applied it in a first pilot study.

Over the last few decades, membrane introduction mass spectrometry (MIMS) has been established as a robust tool for the on-line continuous monitoring of trace gases and volatile organic compounds. However, the range of amenable anlaytes has been limited by the need for molecules to pervaporate into a gaseous acceptor phase, or high vacuum environment of a mass spectrometer. This thesis expands the range of amenable analytes for MIMS to include larger, less volatile molecules (e.g., 200 to 500 Da), such as pharmaceuticals, persistent organic pollutants, and small biomolecules. This was achieved through the use of a liquid|membrane|liquid interface. We distinguish the technique from conventional MIMS, which uses a gaseous acceptor phase, by inserting the prefix ‘condensed phase’ to emphasize the use of a solvent acceptor phase – thus yielding CP-MIMS. An initial flow-cell interface with a methanol acceptor phase was characterized, yielding detection limits for model analytes in pptr to ppb, and analyte response times from 1-10 minutes. The flow cell interface was miniaturized into an immersion style CP-MIMS probe (~2 cm), which allowed for analysis of smaller volume samples and improved membrane washing capabilities. Comparable detection limits were observed for the immersion probe, however, it was noticed that significant analyte depletion was observed for samples under 2 mL. In addition, each of the developed membrane interfaces were observed to suffer from ionization suppression effects from complex samples when paired with ESI. Several strategies for mitigating ionization suppression using CP-MIMS are presented, including the use of a continuously infused internal standard present within the acceptor solvent. The developed CP-MIMS system was challenged with the analysis of naphthenic acids (a complex mixture of aliphatic carboxylic acids) directly in contaminated real-world samples. The method used negative ESI to rapidly screen and mass profile aqueous samples for naphthenic acids (as [M-H]-), with sample duty cycles ~20 min. However, it was found that Negative ESI did not differentiate hydroxylated and carboxylated analytes, and both species contributed signal to the total naphthenic acid concentration. To increase method specificity for carboxylic acids, barium ion chemistry was used in conjunction with positive ion tandem mass spectrometry. Common product ions were used to quantify carboxylated analytes, while a qualifier ion was used to confirm the functionality. The increased selectivity afforded by the barium ion chemistry was at the cost of a modest increase in detection limits. CP-MIMS has been established as a technique capable of the direct analysis of real-world samples, and shows promise as a rapid screening method for amenable environmental contaminants and/or biomolecules. / Graduate / 0486 / 0485 / kyle.duncan@viu.ca

La maladie de Lyme, ou borréliose de Lyme, est une infection bactérienne causée par le spirochète Borrelia burgdorferi sensu lato et transmise à l’hôte (homme, animal) par piqûre de tique du genre Ixodes. Cette maladie, caractérisée par un polymorphisme clinique important, est la maladie à transmission vectorielle la plus répandue dans l’hémisphère nord. Un traitement par antibiotiques permet une guérison rapide, mais si la maladie est diagnostiquée tardivement, certains symptômes persistent. Actuellement, aucun vaccin n’est commercialisé pour l’homme. Dans ce contexte, nous avons développé des approches protéomiques afin d’apporter de nouveaux éléments de compréhension du mécanisme d’interactions de la triade tique / bactérie / hôte. Ces travaux, visant particulièrement le développement de nouvelles stratégies vaccinales et diagnostiques, sont articulés autour de trois parties : - L’identification, suite à de nombreuses optimisations, d’une méthode d’analyse HPLC-UV et nanoLC-MS/MS, de protéines présentes dans des extraits de glandes salivaires de tiques et possédant une activité sur la réponse immunitaire innée cutanée. Ces développements ont mis en évidence une liste restreinte de protéines potentiellement bioactives. - La mise au point, sur un modèle murin, d’une méthode de détection d’une protéine de Borrelia burgdorferi, OspC, dans des biopsies cutanées par spectrométrie de masse ciblée LC-SRM. Cette étude a ouvert des perspectives quant au développement de nouveaux outils diagnostiques. - L’évaluation de la faisabilité de la détection de Borrelia burgdorferi directement à la surface de la peau par imagerie par spectrométrie de masse MALDI-MSI. / Lyme disease, or Lyme borreliosis, is a bacterial infection caused by Borrelia burgdorferi sensu lato and transmitted to the human or animal host by an Ixodes tick bite. This disease, characterized by a huge clinical polymorphism, is the most common vector-born disease in the Northern Hemisphere. An antibiotic treatment allows a fast recovery, but if it is diagnosed too late, some symptoms persist. Currently, no vaccine is marketed for humans. In this context, we have developed proteomic approaches to bring new understanding to the interaction mechanism of the triad tick / bacteria / host. This work, aimed particularly at the development of new vaccinal and diagnostic strategies, has three parts: - Identification, after numerous optimizations, of the analytical method HPLC-UV and nanoLC-MS/MS, of proteins that are present in tick salivary gland extracts and having activity on cutaneous innate immunity response. This work has highlighted a list of proteins with a potential biological activity. - Development, with a murine model, of a method for detecting Borrelia burgdorferi protein, OspC, in cutaneous biopsies by targeted mass spectrometry LC-SRM. This study has opened up perspectives concerning new diagnostic tools. - Evaluation of the feasibility of the Borrelia burgdorferi detection directly on the skin surface by MALDI imaging mass spectrometry.