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Pierce, Carrie. "High throughput mass spectrometry for microbial identification." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/43741.
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Bacteria cause significant morbidity and mortality throughout the world, including deadly diseases such as tuberculosis, meningitis, cholera, and pneumonia. Timely and accurate bacterial identification is critical in areas such as clinical diagnostics, environmental monitoring, food safety, water and air quality assessment, and identification of biological threat agents. At present, there is an established need for high throughput, sensitive, selective, and rapid methods for the detection of pathogenic bacteria, as existing methods, while nominally effective, have failed to sufficiently reduce the massive impact of bacterial contamination and infection. The work presented in this thesis focuses on addressing this need and augmenting conventional microorganism research through development of mass spectrometry (MS)-based proteomic applications. MS, a well established tool for addressing biological problems, offers a broad range of laboratory procedures that can be used for taxonomic classification and identification of microorganisms. These methods provide a powerful complement to many of the widely used molecular biology approaches and play critical functions in various fields of science. While implementation of modern biomolecule-identifying instrumentation, such as MS, has long been postulated to have a role in the microbiology laboratory, it has yet to be accepted on a large scale. Described in this document are MS methods that erect strong foundations on which new bacterial diagnostics may be based. A general introduction on key aspects of this work is presented in Chapter 1, where different approaches for detection of pathogenic bacteria are reviewed, and an overview regarding MS and microbial identification is provided. Chapter 2 presents the first implementation of microbial identification via rapid, open air Direct Analysis in Real Time MS (DART MS) to generate ions directly from microbial samples, including the disease-causing bacteria, Coxiella burnetii, Streptococcus pyogenes, and Escherichia coli. Chapter 3 expands on whole cell C. burnetii MS analysis and presents a rapid differentiation method to the strain-level for C. burnetii using mass profiling/fingerprinting matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and multivariate pattern recognition. Chapter 4 presents a unique "top-down" proteomics approach using 15N-labeled bacteriophage amplification coupled with MALDI-TOF MS as a detector for the rapid and selective identification of Staphylococcus aureus. Chapter 5 extends the idea of using isotopically labeled bacteriophage amplification by implementing a "bottom-up" proteomics approach that not only identifies S. aureus in a sample, but also quantifies the bacterial concentration in the sample using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) as a detector. In conclusion, Chapter 6, summarizes and contextualizes the work presented in this dissertation, and outlines how future research can build upon the experimentation detailed in this document.
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Maple, Hannah Jane. "Towards high-throughput fragment screening by mass spectrometry." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559091.
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Screening for protein-ligand binding interactions is a key step during early stage drug discovery programmes. The fragment-based screening approach has gained popularity in recent years as a highly promising alternative to traditional high-throughput screening (HTS) , which has not yielded the success rate expected in the 'post-genomic' drug discovery era. The increasing use of fragment-based drug discovery (FBDD), however, places additional demands on biophysical screening techniques, and requires that the techniques used are capable of detecting very weak non-covalent interactions (mM Ko values). Existing screerung techniques that have been applied to FBDD, such as nuelear magnetic resonance (NMR), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and X-ray crystallography, are all associated with one or more of the following drawbacks: low throughput, high sample consumption and dynamic range limitations. The use of nano- electrospray mass spectrometry (nano-ESI MS) as a means of screening for non-covalent complexes is a relatively recent addition to existing methodologies that shows promise as an orthogonal screening technique. The advantages it offers: high throughput, low sample consumption, generation of stoichiometric information and the ability to determine dissociation constants make this an attractive approach. Presented here is the development and validation of a fully automated screen by nano-ESI MS, capable of detecting fragment binding into the mM KD range. The method was applied for screening against the anti-apoptotic protein target, Bel-XL, and mass spectrometry results were validated using STD-NMR, HSQC-NMR and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for primary screening. Two alternate, or secondary screerung techniques are also explored. Equilibrium dialysis, combined with nano-ESI MS and STD-NMR, was investigated for the identification of bioactive atropisomers to therapeutic protein targets, Bel-XL and Bcl-2, from mixtures of rotameric compounds. The methodology developed offers an alternative, 'ligand-detect' approach to screening by nano-ESI MS for cases where direct detection of the protein-ligand complex is not possible. Preliminary data are also shown for the application of a microflow capillary NMR probe to automated screening by HSQC NMR experiments. Capillary probes offer excellent mass sensitivity and can be fully automated through interfacing with a liquid handling system. This has the potential to offer a novel fragment screening platform that provides information on the location of compound binding through chemical shift perturbations.
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Boström, Tove. "High-throughput protein analysis using mass spectrometry-based methods." Doctoral thesis, KTH, Proteinteknologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-154513.
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In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers. The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.QC 20141022
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MENG, ZHAOJING. "TOWARDS HIGH-THROUGHPUT ANALYSIS OF RNA USING MASS SPECTROMETRY." University of Cincinnati / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1098054876.
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Abdelrazig, Salah M. A. "Mass spectrometry for high-throughput metabolomics analysis of urine." Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/30600/.
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Direct electrospray ionisation-mass spectrometry (direct ESI-MS), by omitting the chromatographic step, has great potential for application as a high-throughput approach for untargeted urine metabolomics analysis compared to liquid chromatography-mass spectrometry (LC-MS). The rapid development and technical innovations revealed in the field of ambient ionisation MS such as nanoelectrospray ionisation (nanoESI) chip-based infusion and liquid extraction surface analysis mass spectrometry (LESA-MS) suggest that they might be suitable for high-throughput metabolomics analysis. In this thesis, LC-MS and high-throughput direct ESI-MS methods using high resolution orbital trap mass spectrometer were developed and validated for untargeted metabolomics of human urine. Three different direct ESI-MS techniques were explored and compared with LC-MS: flow injection electrospray ionisation-MS (FIE-MS), chip-based infusion and LESA-MS of dried urine spots on a cell culture slide. A high-throughput sample preparation protocol was optimised using in-house artificial urine. Urine samples after consumption of green tea and healthy controls were used as a model to explore the performance and classification ability of the direct ESI-MS. High-throughput data pre-processing and multivariate analysis protocols were established for each method. The developed methods were finally applied for the analysis of clinical urine samples for biomarker discovery and to investigate the metabolic changes in osteoarthritis and malaria. Also, the methods were applied to study the effect of oligofructose diet on the gut microbial community of healthy subjects. The analytical performance of the methods for urine metabolomics was validated using quality control (QC) and principal component analysis (PCA) approaches. Rigorous validation including cross-validation, permutation test, prediction models and area under receiver operating characteristic (ROC) curve (AUC) was performed across the generated datasets using the developed methods. Analysis of green tea urine samples generated 4128, 748, 1064 and 1035 ions from LC-MS, FIE-MS, chip-based infusion and LESA-MS analysis, respectively. A selected set of known green tea metabolites in urine were used to evaluate each method for detection sensitivity. 15 metabolites were found with LC-MS compared to 8, 5 and 6 with FIE-MS, chip-based infusion and LESA, respectively. The developed methods successfully differentiated between the metabolic profiles of osteoarthritis active patients and healthy controls (Q2 0.465 (LC-MS), 0.562 (FIE-MS), 0.472 (chip-based infusion) and 0.493 (LESA-MS)). The altered level of metabolites detected in osteoarthritis patients showed a perturbed activity in TCA cycle, pyruvate metabolism, -oxidation pathway, amino acids and glycerophospholipids metabolism, which may provide evidence of mitochondrial dysfunction, inflammation, oxidative stress, collagen destruction and use of lipolysis as an alternative energy source in the cartilage cells of osteoarthritis patients. FIE-MS, chip-based infusion and LESA-MS increased the analysis throughput and yet they were able to provide 33%, 44% and 44%, respectively, of the LC-MS information, indicating their great potential for diagnostic application in osteoarthritis. Malaria samples datasets generated 9,744 and 576 ions from LC-MS and FIE-MS, respectively. Supervised multivariate analysis using OPLS-DA showed clear separation and clustering of malaria patients from controls in both LC-MS and FIE-MS methods. Cross-validation R2Y and Q2 values obtained by FIE-MS were 0.810 and 0.538, respectively, which are comparable to the values of 0.993 and 0.583 achieved by LC-MS. The sensitivity and specificity were 80% and 77% for LC-MS and FIE-MS, respectively, indicating valid, reliable and comparable results of both methods. With regards to biomarker discovery, altered level of 30 and 17 metabolites were found by LC-MS and FIE-MS, respectively, in the urine of malaria patients compared to healthy controls. Among these metabolites, pipecolic acid, taurine, 1,3-diacetylpropane, N-acetylspermidine and N-acetylputrescine may have the potential of being used as biomarkers of malaria. LC-MS and FIE-MS were able to separate urine samples of healthy subjects on oligofructose diet from controls (specificity/sensitivity 80%/88% (LC-MS) and 71%/64% (FIE-MS)). An altered level of short chain fatty acids (SCFAs), fatty acids and amino acids were observed in urine as a result of oligofructose intake, suggesting an increased population of the health-promoting Bifidobacterium and a decreased Lactobacillus and Enterococcus genera in the colon. In conclusion, the developed direct ESI-MS methods demonstrated the ability to differentiate between inherent types of urine samples in disease and health state. Therefore they are recommended to be used as fast diagnostic tools for clinical urine samples. The developed LC-MS method is necessary when comprehensive biomarker screening is required.
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Li, You. "High throughput mass spectrometry based peptide identification search engine by GPUs." HKBU Institutional Repository, 2015. https://repository.hkbu.edu.hk/etd_oa/261.
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Mass spectrometry (MS)based protein and peptide identification has become a solid method in proteomics. In high-throughput proteomics research, the “shotgun method has been widely applied. Database searching is currently the main method of tandem mass spectrometrybased protein identification in shotgun proteomics. The most widely used traditional search engines search for spectra against a database of identified protein sequences. The search engine is evaluated for its efficiency and effectiveness. With the development of proteomics, both the scale and the complexity of the related data are increasing steadily. As a result, the existing search engines face serious challenges. First, the sizes of protein sequence databases are ever increasing. From IPI.Human.v3.22 to IPI.Human.v3.49, the number of protein sequences has increased by nearly one third. Second, the increasing demand of searches against semispecific or nonspecific peptides results in a search space that is approximately 10 to 100 times larger. Finally, posttranslational modifications (PTMs) produce exponentially more modified peptides. The Unimod database (http://www.unimod.org) currently includes more than 1000 types of PTMs. We analyzed the entire identification workflow and discovered three things. First, most search engines spend 50% to 90% of their total time on the scoring module, the most widely used of which is the spectrum dot product (SDP)based scoring module. Second, nearly half of the scoring operations are redundant, which costs more time but does not increase effectiveness. Third, more than half of the spectra cannot be identified via a database search alone, but the identified spectra have a connection with the unidentified ones, which can be clustered by their distances. Based on the above observations, we designed and implemented a new search engine for protein and peptide identification that includes three key modules. First, a parallel index system, based on GPU, organizes the protein database and the spectra with no redundant data, low search computation complexity, and no limitation of the protein database scale. Second, the graphics processing unit (GPU)based SDP module adopts GPUs to accelerate the most time-consuming step in the process. Third, a k-meansbased spectrum-clustering module classifies the unidentified spectra to the identified spectra for further analysis. As general-purpose high-performance parallel hardware, GPUs are promising platforms for the acceleration of database searches in the protein identification process. We designed a parallel index system that accelerated the entire identification process two to five times with no loss of effectiveness, and achieved around 80% linear speedup effect on the cluster. The index system also can be easily adopted by other search engines. We also designed and implemented a parallel SDP-based scoring module on GPUs that exploits the efficient use of GPU registers and shared memory. A single GPU was 30 to 60 times faster than the central processing unit (CPU)based version. We also implemented our algorithm on a GPU cluster and achieved approximately linear acceleration. In addition, a k-meansbased spectrum-clustering module with GPUs can classify the unidentified spectra to the identified spectra at 20 times the speed of the normal k-means spectrum-clustering algorithm.
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Shi, Wunan. "High-Throughput De Novo Sequencing of Transfer RNAs Using Liquid Chromatography-Tandem Mass Spectrometry." University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1378197247.
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Delabrière, Alexis. "New approaches for processing and annotations of high-throughput metabolomic data obtained by mass spectrometry." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS359/document.
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La métabolomique est une approche de phénotypage présentant des perspectives prometteuses pour le diagnostic et le suivi de plusieurs pathologies. La technique d'observation la plus utilisée en métabolomique est la spectrométrie de masse (MS). Des développements technologiques récents ont considérablement accru la taille et la complexité des données. Cette thèse s'est concentrée sur deux verrous du traitement de ces données, l'extraction de pics des données brutes et l'annotation des spectres. La première partie de la thèse a porté sur le développement d'un nouvel algorithme de détection de pics pour des données d'analyse par injection en flot continue (Flow Injection Analysis ou FIA), une technique haut-débit. Un modèle dérivé de la physique de l'instrument de mesure prenant en compte la saturation de l'appareil a été proposé. Ce modèle inclut notamment un pic commun à tous les métabolites et un phénomène de saturation spécifique pour chaque ion. Ce modèle a permis de créer une workow qui estime ce pic commun sur des signaux peu bruités, puis l'utilise dans un filtre adapté sur tous les signaux. Son efficacité sur des données réelles a été étudiée et il a été montré que proFIA était supérieur aux algorithmes existants, avait une bonne reproductibilité et était très proche des mesures manuelles effectuées par un expert sur plusieurs types d'appareils. La seconde partie de cette thèse a porté sur le développement d'un outil de détection des similarités structurales d'un ensemble de spectre de fragmentation. Pour ce faire une nouvelle représentation sous forme de graphe a été proposée qui ne nécessite pas de connaître la composition atomique du métabolite. Ces graphes sont de plus une représentation naturelle des spectres MS/MS. Certaines propriétés de ces graphes ont ensuite permis de créer un algorithme efficace de détection des sous graphes fréquents (FSM) basé sur la génération d'arbres couvrants de graphes. Cet outil a été testé sur deux jeux de données différents et a prouvé sa vitesse et son interprétabilité comparé aux algorithmes de l'état de l'art. Ces deux algorithmes ont été implémentés dans des package R, proFIA et mineMS2 disponibles à la communautéMetabolomics is a phenotyping approach with promising prospects for the diagnosis and monitoring of several diseases. The most widely used observation technique in metabolomics is mass spectrometry (MS). Recent technological developments have significantly increased the size and complexity of data. This thesis focused on two bottlenecks in the processing of these data, the extraction of peaks from raw data and the annotation of MS/MS spectra. The first part of the thesis focused on the development of a new peak detection algorithm for Flow Injection Analysis (FIA) data, a high-throughput metabolomics technique. A model derived from the physics of the mass spectrometer taking into account the saturation of the instrument has been proposed. This model includes a peak common to all metabolites and a specific saturation phenomenon for each ion. This model has made it possible to create a workflow that estimates the common peak on well-behaved signals, then uses it to perform matched filtration on all signals. Its effectiveness on real data has been studied and it has been shown that proFIA is superior to existing algorithms, has good reproducibility and is very close to manual measurements made by an expert on several types of devices. The second part of this thesis focused on the development of a tool for detecting the structural similarities of a set of fragmentation spectra. To do this, a new graphical representation has been proposed, which does not require the metabolite formula. The graphs are also a natural representation of MS/MS spectra. Some properties of these graphs have then made it possible to create an efficient algorithm for detecting frequent subgraphs (FSM) based on the generation of trees covering graphs. This tool has been tested on two different data sets and has proven its speed and interpretability compared to state-of-the-art algorithms. These two algorithms have been implemented in R, proFIA and mineMS2 packages available to the community
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Emanuele, Vincent A. II. "Advancements in high throughput protein profiling using surface enhanced laser desorption/ionization time of flight mass spectrometry." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/37287.
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Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI)is one of several proteomics technologies that can be used in biomarker discovery studies. Such studies often have the goal of finding protein markers that predict early onset of cancers such as cervical cancer. The reproducibility of SELDI has been shown to be an issue in the literature. There are numerous sources of error in a SELDI experiment starting with sample collection from patients to the signal processing steps used to estimate the protein mass and abundance values present in a sample.
This dissertation is concerned with all aspects of signal processing related to SELDI's use in biomarker discovery projects. In chapter 2, we perform a comprehensive study of the most popular preprocessing algorithms available. Next, in chapter 3, we study the basic statistics of SELDI data acquisition. From here, we propose a quadratic variance measurement model for buffer+matrix only spectra. This model leads us to develop a modified Antoniadis-Sapatinas wavelet denoising algorithm that demonstrates superior performance when compared to MassSpecWavelet, one of the leading techniques for preprocessing SELDI data. In chapter 4, we show that the quadratic variance model 1) extends to real pooled cervical mucus QC data from a clinical study, 2) predicts behavior and reproducibility of peak heights, and 3) finds four times as many reproducible peaks as the vendor-supplied preprocessing programs.
The quadratic variance measurement model for SELDI data is fundamental and promises
to lead to improved techniques for analyzing the data from clinical studies using this instrument.
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Song, Wei. "MASS SPECTROMETRY-BASED HIGH THROUGHPUT APPROACH FOR IDENTIFICATION OF MOLECULAR MODIFICATION OF OXIDATIVE PROCESS IN RESPIRATORY." Cleveland State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1226685494.
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Lobue, Peter. "Towards the Parallel, Accurate, and High-throughput Mapping of RNA Modifications by Liquid Chromatography Tandem Mass Spectrometry." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595005836099446.
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Howard, James W. "The development of mass spectrometry-based methodologies for the high throughput quantitation of peptides in biological matrices." Thesis, Loughborough University, 2018. https://dspace.lboro.ac.uk/2134/32454.
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The aim of this research was the development of mass spectrometry-based methodologies for the high-throughput quantitation of peptides in biological matrices. Glucagon and GLP-1, which are of interest as biomarkers and in the development of therapeutics, were chosen as model peptides. Immunoassays that are traditionally used to quantify these often perform poorly; therefore, necessitating the development of alternative methodologies. Application of mass spectrometry-based methodologies to these analytes has, however, been limited, primarily due to sensitivity challenges, but also due to analytical challenges associated with their endogenous nature and instability in biological matrices. Chapter 2 describes the development and qualification of the first liquid-chromatography coupled tandem mass spectrometry (LC-MS/MS) method for the quantitation of endogenous glucagon from human plasma. A novel 2D extraction procedure was developed to ensure robustness and sensitivity, whilst a novel surrogate matrix quantitation strategy took into account the endogenous nature of the analyte. A lower limit of quantitation (LLOQ) of 25 pg/mL was qualified, which was a considerable improvement over that previously reported in the literature (250 pg/mL) for a LC-MS/MS method. Clinical samples were cross-validated against a conventional radioimmunoassay (RIA), and similar pharmacokinetic (PK) profiles resulted, demonstrating that the methods were complementary. In Chapter 2 glucagon instability in biological matrix was noted. To characterise this further, in Chapter 3 in vitro glucagon metabolites were identified using high-resolution mass spectrometry (HRMS). Metabolites observed by others (glucagon19-29, glucagon3 29 and [pGlu]3glucagon3 29) in alternative matrices were identified, alongside novel metabolites (glucagon20-29 and glucagon21-29). Cross-interference of these metabolites in immunoassays may help to explain their poor performance, whilst knowledge of metabolism may also aid the development of future stabilisation strategies. The method developed in Chapter 2 was refined in Chapter 4 to improve sensitivity, robustness and throughput, and to add GLP-1 as a secondary analyte. The sensitivity achieved (glucagon: 15 pg/mL LLOQ, GLP-1: 25 pg/mL LLOQ) is the highest reported for both peptides for an extraction avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier transition. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between the methods. Differences between the immunoassay results exemplified the need to develop alternative methodologies. The resulting LC-MS/MS method is considered a viable alternative to immunoassays, for the quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1 in human plasma.
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Devenport, Neil A. "Novel methods for the rapid and selective analysis of biological samples using hyphenated ion mobility-mass spectrometry with ambient ionization." Thesis, Loughborough University, 2014. https://dspace.lboro.ac.uk/2134/14287.
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The increased use of mass spectrometry in the clinical setting has led to a demand for high sample throughput. Developments such as ultra high performance liquid chromatography and the ambient ionization techniques enable high sample throughput by reducing chromatographic run times or by removing the requirement for sample preparation and fractionation prior to analysis. This thesis assesses the reproducibility and robustness of these high throughput techniques for the analysis of clinical and pharmaceutical samples by ion mobility-mass spectrometry. The rapid quantitative analysis of the urinary biomarkers of chronic obstructive pulmonary disease, desmosine and isodesmosine has been performed by ultra high performance liquid chromatography combined with ion mobility-mass spectrometry. The determination of health status based on the free unbound fraction rather than the total bound and unbound desmosine and isodesmosine, significantly reduces the time taken in sample preparation. The potential for direct analysis of the urinary metabolites from undeveloped TLC plates using a solvent extraction surface sample probe is demonstrated. The use of a solvent gradient for the extraction separates urinary metabolites from salts and other matrix components and allows fractionation of the sample as a result of differential retention on the undeveloped RP-TLC plate. This separation, combined with ion mobility-mass spectrometry provides a rapid ambient ionization method for urinary profiling. The combination of a thermal desorption probe with extractive electrospray ionization has been applied to the direct detection of a known genotoxic impurity from a surrogate active pharmaceutical ingredient. The volatility of the impurity compared to the matrix, allowed selective thermal desorption of the analyte, which was ionized by extractive electrospray and detected by mass spectrometry. The use of a rapid on-probe derivatisation reaction, combined with thermal desorption is demonstrated for the direct determination of urinary creatinine. The aqueous acylation of creatinine significantly increases the volatility of the analyte enabling separation from the urine matrix and analysis by thermal desorption extractive electrospray combined with ion mobility-mass spectrometry.
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Blacquiere, Johanna M. "New Directions in Catalyst Design and Interrogation: Applications in Dinitrogen Activation and Olefin Metathesis." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/19968.
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A major driving force for development of new catalyst systems is the need for more efficient synthesis of chemical compounds essential to modern life. Catalysts having superior performance offer significant environmental and economic advantages, but their discovery is not trivial. Well-defined, homogeneous catalysts can offer unparalleled understanding of ligand effects, which proves invaluable in directing redesign strategies. This thesis work focuses on the design of ruthenium complexes for applications in dinitrogen activation and olefin metathesis. The complexes developed create new directions in small-molecule activation and asymmetric catalysis by late-metal complexes.
Also examined are the dual challenges, ubiquitous in catalysis, of adequate interrogation of catalyst structure and performance. Insight into both is essential to enable correlation of ligand properties with catalyst activity and/or selectivity. Improved methods for accelerated assessment of catalyst performance are described, which expand high-throughput catalyst screening to encompass parallel acquisition of kinetic data. A final aspect focuses on direct examination of metal complexes, both as isolated species, and under catalytic conditions. Applications of charge-transfer MALDI mass spectrometry to structural elucidation in organometallic chemistry is described, and the technique is employed to gain insight into catalyst decomposition pathways under operating conditions.
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Spiegel, Christopher [Verfasser], Kai [Gutachter] Simons, and Daniel [Gutachter] Müller. "Development of a high-throughput shotgun-mass spectrometry method for qualitative and quantitative analysis of major mammalian brain gangliosides / Christopher Spiegel ; Gutachter: Kai Simons, Daniel Müller." Dresden : Technische Universität Dresden, 2020. http://d-nb.info/1227832923/34.
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van, Oosten Luuk Nico [Verfasser], and Christian D. [Akademischer Betreuer] Klein. "A novel high-throughput and label-free phenotypic drug screening approach: MALDI-TOF mass spectrometry combined with machine learning strategies / Luuk Nico van Oosten ; Betreuer: Christian D. Klein." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1211820904/34.
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Sjödahl, Johan. "Miniaturized Techniques for Protein Analysis." Doctoral thesis, KTH, Chemistry, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3802.
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Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described.
In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied.
In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I.
Key words:Proteins, peptides, proteases, Antarctickrill,Euphausia superba, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.
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Sanvitale, Caroline E. "Investigation of kinase activation in fibrodysplasia ossificans progressiva." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:3ac802e9-a864-4a0d-8e13-f21bcffc957d.
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Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disease resulting in episodic but progressive extraskeletal bone formation. FOP is caused by missense mutations in the cytoplasmic domain of the type I bone morphogenetic protein (BMP) receptor ACVR1, leading to dysregulated activation. Currently there are no available drug treatments and the structural mechanism of mutant activation is still poorly characterised. To address this, a number of BMP and TGFβ receptors, including FOP mutants of ACVR1 were cloned, expressed and purified for both structural and biophysical experiments. The arginine at the site of most recurrent FOP mutation, R206H, is common across all type I receptors except BMPR1A and BMPR1B which have a lysine at this site. The novel structure of BMPR1B differed to wild-type ACVR1 showing some of the conformational changes expected of the active conformation. However, a variety of disease related ACVR1 mutant structures, including ACVR1 R206H, revealed a surprisingly persistent inactive conformation in the kinase domain. Some conformational changes suggestive of activation were observed in the mutant Q207D affecting the ATP pocket, the β4–β5 hairpin and the activation loop. Additionally, the structure of the Q207E mutant showed a slight release of the regulatory glycine-serine rich domain from its inhibitory position. These subtle changes suggest that the mutant inactive conformation is destabilised and potentially more dynamic. In agreement, all of the ACVR1 mutants showed reduced binding to the inhibitory protein FKBP12. However, mutant phosphorylation of the substrate Smad1 was not constitutive, but dependent on the co-expression of the partner ACVR2, consistent with recent evidence from transgenic knock-out mice. A novel 2-aminopyridine inhibitor scaffold with favourable specificity for ACVR1 was identified using a fluorescence-based thermal shift assay. Further derivatives were characterised with improved potency and selectivity. The crystal structures of ACVR1 bound to these inhibitors showed exquisite shape complementarity, contributing to their favourable specificity. This work has increased the understanding of FOP-associated mutant activation and provided a novel starting scaffold for potential drug development.
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Aftab, Obaid. "Towards High-Throughput Phenotypic and Systemic Profiling of in vitro Growing Cell Populations using Label-Free Microscopy and Spectroscopy : Applications in Cancer Pharmacology." Doctoral thesis, Uppsala universitet, Cancerfarmakologi och beräkningsmedicin, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-234565.
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Modern techniques like automated microscopy and spectroscopy now make it possible to study quantitatively, across multiple phenotypic and molecular parameters, how cell populations are affected by different treatments and/or environmental disturbances. As the technology development at the instrument level often is ahead of the data analytical tools and the scientific questions, there is a large and growing need for computational algorithms enabling desired data analysis. These algorithms must have capacity to extract and process quantitative dynamic information about how the cell population is affected by different stimuli with the final goal to transform this information into development of new powerful therapeutic strategies. In particular, there is a great need for automated systems that can facilitate the analysis of massive data streams for label-free methods such as phase contrast microscopy (PCM) imaging and spectroscopy (NMR). Therefore, in this thesis, algorithms for quantitative high-throughput phenotypic and systemic profiling of in vitro growing cell populations via label-free microscopy and spectroscopy are developed and evaluated. First a two-dimensional filter approach for high-throughput screening for drugs inducing autophagy and apoptosis from phase contrast time-lapse microscopy images is studied. Then new methods and applications are presented for label-free extraction and comparison of time-evolving morphological features in phase-contrast time-lapse microscopy images recorded from in vitro growing cell populations. Finally, the use of dynamic morphology and NMR/MS spectra for implementation of a reference database of drug induced changes, analogous to the outstanding mRNA gene expression based Connectivity Map database, is explored. In conclusion, relatively simple computational methods are useful for extraction of very valuable biological and pharmacological information from time-lapse microscopy images and NMR spectroscopy data offering great potential for biomedical applications in general and cancer pharmacology in particular.
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Bourderioux, Matthieu. "Approches protéomiques pour l’analyse des exosomes de liquides biologiques pour la recherche de biomarqueurs." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB102/document.
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Un biomarqueur est une molécule (ou un ensemble de molécules) présente dans l’organisme qui témoigne de l’apparition d’un processus pathologique. Il permet ainsi de dépister une maladie, d’en prédire sa gravité ou encore d’évaluer l’efficacité d’un traitement. Les liquides biologiques représentent des milieux de choix pour la recherche de biomarqueurs en pathologie humaine car leur collection est habituelle dans la prise en charge des patients et moins invasive comparée aux biopsies d’organes ou de tissus. Dans cette thèse, nous nous sommes intéressés plus particulièrement aux exosomes présents dans ces liquides biologiques. Les exosomes sont des nanovésicules dont le diamètre est compris entre 30 et 100 nanomètres. Ils sont sécrétés par tous les types cellulaires et contiennent des protéines cytoplasmiques et membranaires spécifiques de leur cellule d’origine. L’intérêt majeur des exosomes isolés à partir des liquides biologiques, est qu’ils constituent une source de biomarqueurs. Ils peuvent donc être assimilés à une « biopsie liquide ». L’analyse des exosomes pourrait compléter utilement des examens classiques de dépistage, de diagnostic et de suivi d’une pathologie. Dans le cadre de projet de cette thèse, nous avons appliqué des techniques de protéomique à haut débit pour l’analyse des exosomes. Nous nous sommes tout d’abord intéressés à l’analyse du profil protéique des exosomes urinaires dans le contexte de deux pathologies du tractus urinaire : la cystinurie et le cancer du rein. La cystinurie est une néphropathie lithiasique d’origine génétique pour laquelle il y a peu de marqueurs biologiques pouvant prédire son évolution vers l’insuffisance rénale terminale. Nous avons développé une méthode de préparation des exosomes urinaire permettant d’analyser de façon reproductible leurs profils protéiques. Nous avons appliqué cette méthode à huit patients cystinuriques et comparé les résultats aux profils obtenus chez dix sujets sains. Un panel de 38 protéines différentiellement exprimé dans les exosomes des patients a été identifié et en partie validé par Western blot. Concernant le cancer du rein à cellules claires pour lequel le diagnostic nécessite des prélèvements invasifs par biopsie, nous avons analysé les exosomes urinaires de huit patients avant et après néphrectomie. Nous avons ainsi pu mettre en évidence un panel de 25 protéines surexprimées dans les exosomes des patients. Enfin, le dernier volet de cette thèse a été consacré à l’analyse des exosomes du lavage broncho-alvéolaire provenant de patients MV, maladie d’origine génétique qui atteint principalement les poumons. L’analyse des exosomes de lavage broncho-alvéolaire pourrait permettre de donner un éclairage nouveau sur la physiopathologie de la maladie. Nous avons réalisé la comparaison des profils protéiques des exosomes de quatre patients MV, et six patients asthmatiques. L’ensemble des résultats obtenus au cours de cette thèse montre que l’analyse protéomique des exosomes issus de fluides biologiques peut aider la recherche de biomarqueurs diagnostics ou pronostics de maladiesA biomarker is a molecule (or a cluster of molecule) which will reflect the occurrence of a pathological state, giving us the ability to detect a disease, to predict its severity or to assess drug efficiency. Biological fluids are the golden standards for biomarker research in human as they are routinely collected for patients’ follow-up and are less invasive than biopsies. During my PhD, I focused on exosomes that can be found in these biological fluids. Exosomes are nanovesicles with a diameter ranging between 30 and 100 nanometers. Exosomes are secreted by all cell types and harbor cytoplasmic and membranous proteins specific of their cells of origins. One of the major interest of exosomes enriched from biological fluids is that they represent a valuable source of biomarkers. They can be considered as a « liquid biopsy ». Their analysis could complete classical diagnosis and follow-up tools. In this project, we applied high resolution, high throughput proteomic techniques for exosomes analysis. We firstly focused on protein profiles in urinary exosomes in the context of two urinary tract diseases: cystinuria and kidney cancer. Cystinuria is an inherited autosomal recessive disease that is characterized by the formation of cystine stones in the kidneys. To date, there are no markers to predict the evolution toward end stage renal disease. We developed a method to prepare exosomes in order to reproducibly analyze their protein profiles. We applied this method to eight cystinuria patients and compared their profiles to those of ten healthy subjects. A panel of 38 differentially expressed proteins in patients were found and validated by western blots. We also applied this method to patients with clear cell renal cell carcinoma, for which invasive biopsies are necessary for clear diagnosis. We analyzed urinary exosomes form eight patients before and after nephrectomy. We were able to highlight 25 overexpressed proteins in patients’ exosomes. Eventually, the last part of my thesis was dedicated to the analysis of exosomes enriched from bronchoalveolar lavage fluid collected in cystic fibrosis patients, a disease that affects mostly the lungs. Bronchoalveolar lavage fluid exosomes analysis could give a new insight on the mechanisms of this disease. We compared protein profiles in exosomes from four cystic fibrosis patients and six asthmatic patients. The whole point of this work is to show that proteomic analysis of exosomes isolated from biological fluids could become a golden standard for the discovery of diagnosis or prognosis biomarkers
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Duong-Thi, Minh-Dao. "Introducing weak affinity chromatography to drug discovery with focus on fragment screening." Doctoral thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-24642.
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Fragment-based drug discovery is an emerging process that has gained popularity in recent years. The process starts from small molecules called fragments. One major step in fragment-based drug discovery is fragment screening, which is a strategy to screen libraries of small molecules to find hits. The strategy in theory is more efficient than traditional high-throughput screening that works with larger molecules. As fragments intrinsically possess weak affinity to a target, detection techniques of high sensitivity to affinity are required for fragment screening. Furthermore, the use of different screening methods is necessary to improve the likelihood of success in finding suitable fragments. Since no single method can work for all types of screening, there is a demand for new techniques. The aim of this thesis is to introduce weak affinity chromatography (WAC) as a novel technique for fragment screening. WAC is, as the name suggests, an affinity-based liquid chromatographic technique that separates compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds. In this thesis, WAC is studied for fragment screening on two platforms. The first system comprised a 24-channel affinity cartridge that works in cooperation with an eight-needle autosampler and 24 parallel UV detector units. The second system was a standard analytical LC-MS platform that is connected to an affinity column, generally called WAC-MS or affinity LC-MS. The evaluation criteria in studying WAC for fragment screening using these platforms were throughput, affinity determination and ranking, specificity, operational platform characteristics and consumption of target protein and sample. The model target proteins were bovine serum albumin for the first platform, thrombin and trypsin for the latter. Screened fragments were either small molecule drugs, a thrombin-directed collection of compounds, or a general-purpose fragment library. To evaluate WAC for early stages of fragment elaboration, diastereomeric mixtures from a thrombin-directed synthesis project were screened. Although both analytical platforms can be used for fragment screening, WAC-MS shows more useful features due to easy access to the screening platform, higher throughput and ability to analyze mixtures. Affinity data from WAC are in good correlation with IC50 values from enzyme assay experiments. The possibility to distinguish specific from non- specific interactions plays an important role in the interpretation of WAC results. In this thesis, this was achieved by inhibiting the active site of the target protein to measure off-site interactions. WAC proves to be a sensitive, robust, moderate in cost and easy to access technique for fragment screening, and can also be useful in the early stages of fragment evolution. In conclusion, this thesis has demonstrated the proof of principle of using WAC as a new tool to monitor affinity and to select hits in fragment-based drug discovery. This thesis has indicated the primary possibilities, advantages as well as the limitations of WAC in fragment screening procedures. In the future, WAC should be evaluated on other targets and fragment libraries in order to realize more fully the potential of the technology.
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Oteng, Eugene K. "Discovery of a conserved Plasmodium antigen on the surface of malaria-infected red blood cells." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:592769fc-2628-4c6e-9a68-99060fb8c091.
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During its intraerythrocytic stages (IE), Plasmodium falciparum, the causative agent of the deadliest human malaria, remodels the host red cell membrane with a poorly defined assortment of parasite-encoded proteins that undergo antigenic variation. Despite the requirement for immunologic stealth, exported parasite proteins also mediate strain-independent functions such as endothelial sequestration that are critical for parasite survival and pathogenesis. This thesis explores the hypothesis that P. falciparum displays novel structurally conserved proteins on the IE surface and these proteins may serve as useful antigens for a broadly effective anti-malarial vaccine. In order to test this hypothesis, we developed an in vitro selection technique that sequentially incorporates unique P. falciparum isolates as the targets for Systematic Evolution of Ligands by EXponential enrichment (Serial-SELEX) to generate nucleic acid molecular probes, aptamers, capable of recognizing conserved cell surface determinants. Ten of 11 enriched aptamers were -parasite selective and three of these aptamers demonstrated strain-independent binding to P. falciparum. Aptamer recognition extended beyond the parasites used in Serial-SELEX to other laboratory and recent field isolates. Surprisingly the same three broadly binding aptamer selected against P. falciparum also recognized all laboratory-adapted and clinical isolates of P. vivax and P. knowlesi tested, strongly supporting our hypothesis that structurally conserved molecules are present on the surface IEs. Competition studies showed that the aptamers bound a single target which was confirmed as an IE membrane protein. Aptamer‐mediated affinity purification and tandem mass spectrometry enabled identification of the aptamer target as parasite-encoded protein. Discovery of a protein conserved between the major human malarias may have implications for vaccine development and validates the Serial‑SELEX technique as a powerful tool for antigen discovery.
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Jouve, Thomas. "Étude des déterminants de la puissance statistique en spectrométrie de masse." Phd thesis, Université Claude Bernard - Lyon I, 2009. http://tel.archives-ouvertes.fr/tel-00635493.
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La spectrométrie de masse fait partie des technologies haut débit et offre à ce titre un regard inédit, à une échelle nouvelle, sur les protéines contenues dans divers échantillons biologiques. Les études biomédicales utilisant cette technologie sont de plus en plus nombreuses et visent à détecter de nouveaux biomarqueurs de différents processus biologiques, notamment de processus pathologiques à l'origine de cancers. Cette utilisation comme outil de criblage pose des questions quant à la capacité même des expériences de spectrométrie de masse dans cette détection. La puissance statistique traduit cette capacité et rappelle que les études doivent être calibrées pour offrir des garanties suffisantes de succès. Toutefois, cette exploration de la puissance statistique en spectrométrie de masse n'a pas encore été réalisée. L'objet de cette thèse est précisément l'étude des déterminants de la puissance pour la détection de biomarqueurs en spectrométrie de masse. Une revue de la littérature a été réalisée, reprenant l'ensemble des étapes nécessaires du traitement du signal, afin de bien comprendre les techniques utilisées. Les méthodes statistiques disponibles pour l'analyse du signal ainsi traité sont revues et mises en perspective. Les situations de tests multiples, qui émergent notamment de ces données de spectrométrie de masse, suggèrent une redéfinition de la puissance, détaillée par la suite. La puissance statistique dépend du plan d'expérience. La taille d'échantillon, la répartition entre groupes étudiés et l'effet différentiel ont été investigués, par l'intermédiaire de simulations d'expériences de spectrométrie de masse. On retrouve ainsi les résultats classiques de la puissance, faisant notamment ressortir le besoin crucial d'augmenter la tailles des études pour détecter des biomarqueurs, particulièrement lorsque ceux-ci présentent un faible effet différentiel. Au delà de ces déterminants classiques de la puissance, des déterminants propres à la spectrométrie de masse apparaissent. Une chute importante de puissance est mise en évidence, due à l'erreur de mesure des technologies de spectrométrie de masse. Une synergie péjorative existe de plus entre erreur de mesure et procédure de contrôle du risque de première espèce de type FDR. D'autre part, les méthodes de détection des pics, par leurs imperfections (faux pics et pics manqués), induisent un contrôle suboptimal de ce risque de première espèce, conduisant à une autre chute de puissance. Ce travail de thèse met ainsi en évidence trois niveaux d'intervention possibles pour améliorer la puissance des études : la meilleure calibration des plans d'expérience, la minimisation de l'erreur de mesure et l'amélioration des algorithmes de prétraitement. La technologie même de spectrométrie de masse ne pourra conduire de façon fiable à la détection de nouveaux biomarqueurs qu'au prix d'un travail à ces trois niveaux.
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Habchi, Baninia. "Mise en évidence des perturbations métaboliques liées à l’exposition aux toxiques présents dans l’environnement ou l’aliment par spectrométrie de masse à ultra haute résolution FTMS combinée avec des outils chimiométriques." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLA032.
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The implementation of genomic selection makes possible the inclusion of new traits in breeding goals, by taking advantage of the opportunities coming from the increased genetic trend on traits currently under selection. Breeders, breeding companies and society all have changing expectations regarding genetic selection. Two groups of new traits were analyzed in the context of genetic improvement of dairy cattle: carcass traits of young bulls in dual-purpose breeds and claw health traits in Holstein, in order to prepare the implementation of genetic and genomic selection on these traits. For both sets of traits, suitable genetic evaluation models were developed and genetic parameters were estimated. Genetic parameters reveal that genetic selection of carcass traits of young bulls appears to be fairly easy and that selection of claw health traits is going to be more difficult, but possible, given the existing genetic variation. They also highlight that there is no strong negative genetic correlation between carcass traits of young bulls and dairy production traits. Finally, they reveal that there are two genetically distinct groups of claw health traits. Several strategies to account for non-exhaustive recording of cows for trimming were tested. Several evaluation approaches were compared. For both sets of traits, Single-Step Genomic BLUP was the most promising approach, although other (two-step) genomic approaches allowed for relatively similar accuracies and control of bias. These studies led to the implementation of routine genetic and genomic evaluations for both sets of traits, for which a usual two-step genomic approach was preferred over Single-Step Genomic BLUP for consistency with the current evaluation of other traits. However these two examples illustrate the benefit of implementing routine Single-Step Genomic BLUP evaluations. The main questions and principal steps identified in these studies were gathered into tentative guidelines for the development of genetic evaluations for new traits. d‘un grand nombre d‘échantillons biologiques pour mettre en évidence les perturbations métabolomiques induites par l‘exposition à des substances toxiques. Dans une première partie de ce travail, nous nous sommes intéressés à l‘étude d‘un petit groupe d‘agriculteurs ayant été exposés dans le cadre professionnel à deux pesticides. Une mise au point de l‘approche DIMS sur un instrument de type Orbitrap a été réalisée. Une procédure de traitement des données a aussi été développée. Elle est basée sur un nouvel outil dite IC-DA (independent component - discriminant analysis). Cette méthodologie a ensuite été appliquée sur un nombre d'échantillons plus élevé issus de cinq types d‘exposition. Dans cette étude, deux approches analytiques DIMS et LC/MS ont été examinées afin de valider l‘approche DIMS mais aussi l‘outil de traitement chimiométrique développé. Une deuxième partie est dédiée aux travaux visant à étendre l‘approche à haut débit sur un instrument de performances plus élevées, le FT-ICR (Fourier transform - ion cyclotron resonance) équipé d'une nouvelle cellule de piégeage dite ̎dynamically harmonized cell ̎. Une première étude a concerné l‘exposition des rats à différents concentration de pesticides. Dans une deuxième étape, une étude sur un nombre important d‘échantillon d‘urines humaines (d‘environ 500 individus) a été réalisée pour tester la robustesse de notre approche. Mes travaux ont permis de démontrer la faisabilité et l'efficacité de notre approche métabolomique à haut débit combinant l‘introduction directe DIMS, la détection à très haute résolution et les outils chimiométriques. Cette approche pourrait être très prometteuse pour réaliser le phénotypage métabolique à grande échelle, comme dans les études épidémiologiquesPublic health monitoring involves evaluation of population exposure to environmental toxicants which can have an impact on their health. To do this, robust and high-throughput approaches are required to perform large scale analyses. Global approaches such as metabolomics which aim to reveal metabolic changes due to environmental stress or diseases seem to be the most appropriate approach. This multidisciplinary approach requires powerful analytical techniques such as mass spectrometry (MS) associated with statistical and chemometric data processing. It allows to detect general metabolic disruptions induced by a given physiological or pathological conditions. The studied samples can be injected either directly by the DIMS technique (direct introduction mass spectrometry) or following a chromatographic separation using GC/MS or LC/MS (gas or liquid chromatography / mass spectrometry). The DIMS approach leads to a significant reduction in analysis time, down to only a few minutes (usually less than 3 min). Additionally, in combination with Fourier transform mass spectrometers (DIFTMS), it provides very high mass resolving power and accurate mass measurements, as well as a wide dynamic range resulting in improved efficiency. Nevertheless, the DI-FTMS approach generates complex data containing several thousands of peaks. Processing such large data sets requires the development of dedicated chemometric and statistical tools to detect exposure biomarkers. Therefore, the objective of my work was to develop a rapid, highthroughput workflow, including the development of chemometric tools, in order to highlight metabolomic perturbations induced by exposure to toxicants. The first part of this work concerns the study of farmers professionally exposed to two pesticides. The DIMS approach was performed on an Orbitrap instrument and a new chemometric tool called Independent Component - Discriminant Analysis (IC-DA) was developed for supervised analysis of the DIMS data. The developed methodology was then applied to a larger number of samples corresponding to five types of exposure. In this later study, two analytical approaches DIMS and LC/MS were examined in order to validate the DIMS approach as well as the developed chemometric data analysis tool. In a second part of this work, the DIMS approach was applied to an instrument of higher performances, the FT-ICR (Fourier transform-ion cyclotron resonance) equipped with a dynamically harmonized cell in order to improve the quality of the DIMS data. A first study explored the effects of exposure of rats to different concentrations of pesticides. In a second step, the procedure was applied to a large number of samples (of approximately 500 individuals) to test the robustness of the approach. All this work demonstrated the feasibility and effectiveness of our high-throughput metabolomic approach combining the direct introduction (DIMS), the very high resolution detection and the chemometric tools. This approach could be very promising to perform large scale metabolic phenotyping such as in epidemiological studies
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Largy, Eric. "Ciblage d’acides nucléiques G-quadruplexes : synthèse et développement de méthodes pour l’analyse et le criblage de ligands sélectifs multimodaux." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112257.
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L’objectif de ces travaux de thèse était l’étude des interactions de petites molécules avec les multiples structures de l’ADN quadruplex via i) le développement et l’utilisation d’un test haut-débit pour l’analyse des interactions ligand-ADN quadruplex et le criblage de chimiothèques/ciblothèques et ii) la préparation de composés aux modes d’interactions multiples (empilement/sillon, covalent/non-covalent, etc.), sélectifs (quadruplex vs. duplex et intra-quadruplex) et éventuellement fonctionnalisés (biotine, fluorophore, etc.). La première partie des travaux a été centrée sur le développement du test G4-FID (G-quadruplex Fluorescent Intercalator Displacement) qui est une méthode semi-quantitative permettant l’évaluation de l’affinité et de la sélectivité de petites molécules pour l’ADN quadruplex par déplacement d’une sonde off/on, le Thiazole Orange (TO). Le test a notamment été transposé avec succès de la cuve vers la microplaque (HT-G4-FID). D’autre part, nous avons montré l’intérêt de fluorophores alternatifs, TO-PRO-3 et Hoechst 33258, aux caractéristiques spectrales complémentaires à TO. Cette méthode d’analyse a également été utilisée avec succès pour l’identification de nouveaux ligands sélectifs d’ADN quadruplex et la mise en évidence des relations structure-activité ainsi que des sélectivités structurales. La deuxième partie des travaux a été consacrée à la préparation et à l’étude de nouveaux ligands d’ADN quadruplex. Ces ligands possèdent des particularités, soit dans leur mode d’interaction (sillons, coordination) soit par leur bifonctionnalité (biotinylés, fluorescents). Nous avons ainsi préparé un ligand de quadruplex polyhétéroaryle acyclique (TOxaPy) possédant une sélectivité inattendue pour certaines structures de l’ADN quadruplex. D’autre part, nous avons montré que les complexes de dérivés de terpyridine peuvent être adaptés, en changeant le ligand organique et/ou la nature du métal, de façon à interagir avec l’ADN quadruplex par interaction covalentes et/ou non covalentesThe aim of this thesis work was to study the interactions of small molecules with multiple structures of quadruplex DNA via i) the development and use of a high-throughput test for the analysis of ligand-quadruplex DNA interactions and screening of chemical libraries and ii) the preparation of compounds with multiple binding modes (stacking/groove, covalent/non-covalent, etc..) selective (quadruplex vs. duplex and intra-quadruplex) and possibly functionalized (biotin, fluorophore, etc.). The first part of the work was focused on the development of the G4-FID (G-quadruplex Intercalator Fluorescent Displacement) assay, which is a semi-quantitative method for evaluating the affinity and selectivity of small molecules for quadruplex DNA by displacing an off/on probe, the Thiazole Orange (TO). The test has been implemented successfully with microplate (HT-G4-FID). On the other hand, we have shown the importance of alternative fluorophores, TO-PRO-3 and Hoechst 33258, with complementary spectral characteristics. This method of analysis has also been successfully used for the identification of new selective ligands of quadruplex DNA and the identification of structure-activity relationships and structural selectivities. The second part of the work was devoted to the preparation and study of new DNA quadruplex ligands. These ligands possess particular characteristics either in their mode of interaction (grooves, coordination) or by their bifunctionality (biotinylated, fluorescent). We have prepared an acyclic polyheteroaryle quadruplex ligand (TOxaPy) with an unexpected selectivity for certain structures of quadruplex DNA. Furthermore, we showed that complexes of terpyridine derivatives can be tailored by changing the organic ligand and / or the metal in order to interact with quadruplex DNA by covalent and / or non-covalent interaction
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Stoppe, Nancy de Castro 1963. "Prospecção de marcadores para o rastreamento de fontes de contaminação fecal em águas superficiais do Estado de São Paulo." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316933.
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Orientadores: Laura Maria Mariscal Ottoboni, Tatiana Teixeira TorresTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A contaminação fecal dos corpos hídricos é uma das principais causas de doenças entéricas veiculadas pela água no mundo, sendo importante efetuar a vigilância da água, a qual é feita utilizando-se micro-organismos indicadores de contaminação fecal. No entanto, os métodos tradicionais de detecção não são capazes de identificar a fonte de contaminação fecal. Este trabalho teve como objetivo a prospecção de marcadores moleculares nos hospedeiros e sua detecção em amostras de água de modo a permitir sua utilização na identificação de fontes de contaminação fecal em águas superficiais no Estado de São Paulo. Duas abordagens dependentes de biblioteca e cultivo, a classificação por meio de grupos filogenéticos e a técnica de MALDI-TOF/MS, foram utilizadas com linhagens de E. coli isoladas de diferentes hospedeiros e rios e reservatórios. O sequenciamento da região V3 do gene 16S ribossomal foi selecionado como o método independente de biblioteca e cultivo em DNA extraído de amostras de fezes humanas e bovinas e amostras de água. Os grupos filogenéticos foram utilizados na classificação dos hospedeiros utilizando análise de correspondência, onde foram observados agrupamentos por hábitos alimentares. A classificação das linhagens de E. coli isoladas de rios e reservatórios, sugere que a prevalência do subgrupo A1, seguido do subgrupo B23 está associada com contaminação de origem humana, enquanto o grupo B1 com contaminação de origem animal e os subgrupos D1 e D2 mais relacionados com ambientes prístinos. Na utilização de uma métrica de análise de rede social, w-clique, na distribuição dos grupos filogenéticos foi observado o agrupamento dos locais de amostragem associados ao grau de poluição, sugerindo seu uso como uma ferramenta complementar na avaliação da qualidade da água. Foram analisados os perfis proteicos de linhagens de E. coli de hospedeiros e amostras ambientais pela técnica de MALDI-TOF/MS. Foram identificados biomarcadores hospedeiro-específicos e sugerem sua utilização como potencial ferramenta na identificação da origem do hospedeiro. Os resultados da validação desses marcadores com perfis proteicos de linhagens de E. coli isoladas de rios e reservatórios mostraram que as amostras de água apresentaram marcadores de diferentes hospedeiros, sugerindo que esses rios possuam fontes de contaminação fecal mistas. No sequenciamento da região V3 do gene 16S ribossomal de amostras de fezes (humanas e bovinas) e águas foram identificadas 4.296 unidades taxonômicas operacionais (OTUs) . A maior diversidade foi observada nas amostras de fezes bovinas e a menor na amostra de água de ambiente prístino. Firmicutes foi o grupo predominante nas amostras de fezes humanas, enquanto que nas fezes bovinas foram os Firmicutes e Bacteroidetes. Nas amostras de água, o filo mais abundante foi Proteobacteria. A rede de interação entre as OTUs encontradas nas amostras também mostrou que as amostras de fezes apresentaram maior diversidade e entre as amostras de água, aquela com poluição de origem humana foi a que apresentou maior diversidade. Houve identificação de biomarcadores pelo método LEfSe para humanos (Actinobacteria, Betaproteobacteria e Firmicutes) e bovinos: (Bacteroidetes, Tenericutes e Spirochaetes). Marcadores hospedeiro-específicos foram identificados, mas esses não foram encontrados nas amostras de água sugerindo que as ferramentas utilizadas não apresentam a resolução para identificar os marcadores nas amostras ambientais ou que a contaminação nos corpos hídricos é mista. Adicionalmente, como os marcadores hospedeiro-específicos são oriundos de micro-organismos não autóctones, estes poderiam sofrer os efeitos adversos do ambiente, como fatores físico-químicos e competição com os organismos nativos
Abstract: The fecal contamination of water resources is the main cause of enteric waterborne diseases all over the world. Traditional indicator methods used in the water microbiological quality assessment are not able to identify fecal contamination source. This work intended to prospect molecular markers in hosts and track them in water samples to identify pollution sources in surface waters in the São Paulo State, Brazil. Two library-dependent methods with E. coli strains isolated from different hosts and water samples were used, a genotypic typing method (E. coli phylogenetic groups) and a phenotypic typing method (MALDI-TOF/MS). A library-independent method using 454 pyrosequencing of hypervariable16S rRNA gene V3 region was used in DNA from feces and water samples. Phylogenetic groups were used as a tool in host classification and correspondence analysis showed feeding habits clusters. The classification of environmental samples revealed higher frequencies of subgroups A1 and B23 in rivers impacted by human pollution sources, while subgroups D1 and D2 were associated with pristine sites, and subgroup B1 with domestic animal sources, indicating their use as a first screening for pollution source identification. A simple classification is proposed based on phylogenetic subgroup distribution using the w-clique metric, enabling differentiation of polluted and unpolluted sites. Protein profiles of E. coli strains isolated from host and water samples were analyzed by MALDI-TOF/MS. Specific host biomarkers were identified and their use was indicated as a potential tool for the source tracking. Validation with E. coli strains isolated from rivers and reservoirs showed that water samples presented markers from different hosts, suggesting these rivers have mixed sources of fecal contamination. Sequencing of the 16S rRNA V3 region in stool samples (human and bovine) and water showed 4296 operational taxonomic units (OTUs). The greatest diversity was observed in samples of cattle feces and the smallest one in the pristine water sample. Firmicutes was the predominant group in samples of human feces, while in the most common bovine feces are the Firmicutes and Bacteroidetes. The interaction network showed that the stool samples had the greatest diversity and, among them, the water sample with human pollution source showed the highest diversity. The LEfSe method was used to identify host biomarkers. As human biomarkers, Actinobacteria, Betaproteobacteria and Firmicutes were identified and for cattle the potential markers are Bacteroidetes, Tenericutes and Spirochaetes. Host-specific markers were identified, but they were not found in water samples suggesting that the used tools either do not have the resolution to identify markers in environmental samples or contamination in water bodies is mixed. Additionally, as the host-specific markers were isolated from non-autochthonous micro-organisms, they could be affected by the environmental adverse effects such as physical-chemical factors and competition with native organisms
Doutorado
Microbiologia
Doutora em Genética e Biologia Molecular
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(8086205), David L. Logsdon. "HIGH-THROUGHPUT ORGANIC REACTION SCREENING USING DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY." Thesis, 2019.
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This dissertation describes the development of a system for the automated, high-throughput screening of organic reactions. This system utilizes a liquid handling robot for reaction mixture preparation combined with desorption electrospray ionization mass spectrometry (DESI-MS) for reaction mixture analysis. With an analysis speed of ~1 second per reaction mixture, this system is capable of screening thousands of reactions per hour. Reaction mixtures are prepared in 384-well microtiter plates using a liquid handling robot. A sample of each reaction mixture (50 nL) is then transferred to a PTFE coated, glass slide using a pin tool. By offsetting the placement of the pin tool during each transfer, up to 6,144 unique reaction mixtures can be placed on each slide. The slide is then transferred to the DESI stage by a robotic arm, and the DESI-MS analysis begins, taking as little as 7 minutes for 384 reaction mixtures. We utilize a scheduling software to control each component of the system, which automates the entire process from reaction mixture preparation to DESI-MS analysis. In order to efficiently analyze and visualize the extremely large data sets generated by the system, we developed a custom software suite to automatically process each data set. We have used this system to screen several classes of industrially relevant reactions including Suzuki coupling, nucleophilic aromatic substitution, reductive amination, and Sonogashira coupling. We have validated both positive and negative results from the system using flow chemistry, and we have observed excellent agreement between the two methodologies. By being capable of screening thousands of reactions per hour, requiring only microliter quantities of reaction mixtures, and consuming less than a milliliter of solvent during the DESI-MS analysis, this system significantly reduces the time and costs associated with organic reaction screening.
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LeDuc, Richard D. "Bioinformatics of high throughput proteomics using tandem mass spectrometry of intact proteins /." 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3290288.
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Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7046. Adviser: Gustavo Caetano-Anolles. Includes bibliographical references (leaves 117-129) Available on microfilm from Pro Quest Information and Learning.
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Boltz, Stacey A. "High throughput proteomic studies using fourier transform ion cyclotron resonance mass spectrometry." 2003. http://purl.galileo.usg.edu/uga%5Fetd/boltz%5Fstacey%5Fa%5F200305%5Fms.
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Chen, Ming-Shou, and 陳明壽. "Biomarker discovery on high-throughput and high-resolution mass spectrometry oral cancer data using statistical methods." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/56cbf4.
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碩士國立中央大學
資訊工程研究所
94
Due to the high death rate in advanced stage diseases, the diagnosis of early-stage cancer is needed for public health. Recent advances in the biotechnology of high-throughput and high-resolution MALDI-TOF mass spectrometry (MS) has made such diagnosis possible (Petricoin and Liotta 2003). From then on, high-resolution mass spectrometers are increasingly used for disease classification and therapeutic guidance. Due to instrument resolution and/or instrument calibration, the mass spectrometry (MS) data may be poor in quality. The problem makes it difficult and time consuming to trace each spectrum with thousands of features for biomarkers. We proposed a region-based alignment method to deal with peak shifting problem and applied statistical method to select most discriminatory peaks as biomarker candidates. Finally, we test our methodology on the oral cancer dataset from CHANG GUNG UNIVERSITY in Taiwan.
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(7900775), Harrison S. Ewan. "HIGH THROUGHPUT EXPERIMENTATION WITH DESORPTION ELECTROSPRAY IONIZATION MASS SPECTROMETRY TO GUIDE CONTINUOUS-FLOW SYNTHESIS." Thesis, 2019.
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The present work seeks to use high throughput experimentation (HTE) to guide chemical synthesis. We demonstrate the use of an HTE system utilizing a robotic liquid handler to prepare arrays of reactions and print them onto a surface to be analyzed by desorption electrospray ionization mass spectrometry (DESI-MS) as a tool to guide reaction optimization, synthetic route selection, and reaction discovery. DESI-MS was employed as a high throughput experimentation tool to provide qualitative predictions of the outcome of a reaction, so that vast regions of chemical reactivity space may be more rapidly explored and areas of optimal efficiency identified. This work is part of a larger effort to accelerate reaction optimization to enable the rapid development of continuous-flow syntheses of small molecules in high yield. In the present iteration of this system, reactions are scaled up from these nanogram surface printed reactions to milligram scale microfluidic reactions, where more detailed analysis and further optimization may be performed. In the earliest iterations of this screening system prior to the use of DESI, the initial screening reactions were performed in electrospray (ESI) droplets and leidenfrost droplets before scaling up to microfluidic reactions which were analyzed by ESI-MS. The insights from this combined droplet and microfluidic screening/rapid ESI-MS analysis approach, helped guide the synthesis of diazepam. The system was further refined to by the use of liquid handling robots and DESI-MS analysis, greatly accelerating the overall pace of screening. In order to build confidence in this approach, however, it is necessary to establish a robust predictive connection between reactions performed under analogous DESI-MS, batch, and microfluidic reaction conditions. To achieve this goal, we first explored the potential of high throughput DESI-MS experiments to identify trends in reactivity based on chemical structure, solvent, temperature, and stoichiometry that are consistent across these platforms. While DESI-MS narrowed the scope of possibilities for reaction
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selection with some parameters such as solvent, others like stoichiometry and temperature still required further optimization under continuous synthesis conditions. With our increased confidence in DESI-MS HTE, we proceeded to explore it’s application to rapidly evaluate large sets of aldol reactions of triacetic acid lactone (TAL), a compound well studied for use as a bio-based platform molecule that may be converted to a range of useful commodity chemicals, agrochemicals, and advanced pharmaceutical intermediates. Our DESI-MS HTE screening technique was used to rapidly evaluate known reactions of triacetic acid lactone, in an effort to accelerate reaction discovery with platform chemicals. Our rapid experimentation system, when applied to reaction discovery in this manner, may help to shorten the time scale of platform chemical development.
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"The development of high-throughput mass spectrometric methods for the qualitative and quantitative analysis of diquaternary ammonium gemini surfactants." Thesis, 2013. http://hdl.handle.net/10388/ETD-2013-11-1289.
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For over a decade, diquaternary ammonium gemini surfactants have shown promise as non-viral gene delivery agents in both in vitro and in vivo systems. Their continued development, however, requires an understanding of their biological fate. The absence of identification and quantification methods that can achieve that goal is what drove the development of simple and rapid mass spectrometry (MS)-based methods; the focus of my Ph.D. dissertation.
Prior to the development of these MS-based methods, an understanding of the gas phase behavior of diquaternary ammonium gemini surfactants is required. The development of a universal fragmentation pathway for gemini surfactants was achieved using low resolution and high resolution MS instruments. Single stage (MS), tandem stage (MS/MS and quasi-multi-stage (quasi MS3) mass spectrometry analysis allowed for the confirmation of the molecular composition and structure of each gemini surfactant through the identification of common and unique mass to charge values. Understanding the fragmentation behavior allowed for the specific identification and/or quantification of gemini surfactants by MS-based methods; including liquid chromatography low resolution tandem mass spectrometry (LC-LR-MS/MS), fast chromatography low resolution tandem mass spectrometry, fast chromatography high resolution mass spectrometry, desorption electrospray ionization low resolution mass spectrometry and matrix assisted laser desorption ionization high resolution mass spectrometry.
We hypothesized that a LC-LR-MS/MS method would be the most effective quantitative method for the quantification of N,N-bis(dimethylhexadecyl)-1,3-propane-diammonium dibromide (G16-3) within PAM212 cellular lysate; achieving the lowest lower limit of quantification (LLOQ). Although the LC-LR-MS/MS method achieved a LLOQ suitable for analysis of G16-3 within PAM212 cell lysate, its limitations made it an inefficient method. In comparison, the four alternative mass spectrometry methods were faster, more efficient and less expensive than a conventional LC-LR-MS/MS method for the post transfection quantification of G16-3 within PAM212 cell lysate to be determined; 1.45 ± 0.06 μM. Future application of the universal fragmentation pathway and each MS-based quantification method will be beneficial for the future development of diquaternary ammonium gemini surfactants to further understand their post transfection fate.
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Chang, Yuan-Jhe, and 張元哲. "Method Development of the New Generation Hair Testing by High Throughput Liquid Chromatography Tandem Mass Spectrometry." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/52018740213623060734.
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博士中山醫學大學
醫學研究所
102
Compared to blood and urine, hair is unique in that it could determine the time period of chemical exposure after several months to years. The main purpose of this study is to develop high throughput and sensitivity analysis method of hair testing. Firstly, we developed a fast microwave-assisted extraction method for reducing the incubation time of abused drug release from human hair. In addition, extraction-free sample preparation and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis are evaluated for improving throughput of experiments. Secondly, a high sensitive method for detecting trace amount THC-COOH in hair by using strategy of simple and fast chemical derivatization with dansyl chloride coupled with LC–MS/MS. Thirdly, a stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to detect the metabolites of human DEHP exposure in hair. We investigated the incubation conditions for improving the releae efficiency of metabolites from hair. Furthermore, an automated on line solid-phase extraction (On-line SPE) coupled with LC–MS/MS system was developed for reducing experiment time and increasing the high-throughput of analysis. In particular, the authentic hair specimens were determined and perfromed of segmental hair analysis for proving the practicability of assessing DEHP exposure in human hair. Consequently, an LC–MS/MS method was developed and utilized to authentic hair that could be successfully determined of parent compound of clobenzorex to discriminate the truly condition of clobenzorex used while not AMP.
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Lin, Shiau-Pei, and 林曉珮. "High Throughput Screening for Zearalenone by Matrix-Assisted Laser Desorption/Inoization Time-of -Flight Mass Spectrometry." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/24451532426214919195.
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碩士朝陽科技大學
應用化學系碩士班
98
Zearalenone (ZEA) is one of the mycotoxins which are the secondary metabolites of fungi. It is found in corn, wheat, barley and oats. In Taiwan, the official analysis method for ZEA is using disposable immunoaffinity column coupling with high performance liquid chromatography (HPLC) which has the detection limit of 10 ppb. However, the modern preservation of grain has reduced the contamination of ZEA in corps. The cost of the specific column and the time consuming are not good for high throughput analysis. If there is a low cost and high throughput screening methods to filter out most uncontaminated samples, a lot of time consuming and cost would be avoided. Here we report a low cost and high throughput screening method for the analysis of ZEA using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method includes two parts. First, the sample preparation using celite to remove sample matrix is simple and low cost. Second, without general matrix for co-crystal with analyte, aluminum foil is used to obtain low noise analyte spectra in low mass range. The cost of sample preparation for each sample is less than NT$ 10.0 dollars, and analysis time for each sample is only 1 min or less, detection limit can reach to 10 ppb which is comparable with the official method.
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(9137873), Zhuoer Xie. "Accelerating the Throughput of Mass Spectrometry Analysis by Advanced Workflow and Instrumentation." Thesis, 2020.
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The exploratory profiling and quantitative bioassays of lipids, small metabolites, and peptides have always been challenging tasks. The most popular instrument platform deployed to solve these problems is chromatography coupled with mass spectrometry. However, it requires large amounts of instrument time, intensive labor, and frequent maintenance, and usually produces results with bias. Thus, the pace of exploratory research is one of poor efficacy and low throughput. The work in this dissertation provides two practical tactics to address these problems. The first solution is multiple reaction monitoring profiling (MRM-profiling), a new concept intended to shift the exploratory research from current identification-centered metabolomics and lipidomics to functional group screening by taking advantage of precursor ion scan and product ion scan. It is also demonstrated that MRM-profiling is capable of quantifying the relative amount of lipids within the same subclass. Besides, an application of the whole workflow to investigate the strain-level differences of bacteria is described. The results have zeroed in on several potential lipid biomarkers and corresponding MRM transitions. The second strategy is aimed to increase the throughput of targeted bioassays by conducting induced nanoelectrospray ionization (nESI) in batch mode. A novel prototype instrument named "Dip-and-Go" system is presented. Characterization of its ability to carry out reaction screening and bioassays exhibits the versatility of the system. The distinct electrophoretic cleaning mechanism contributes to the removal of salt during ionization, which assures the accuracy of measurement.
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Wang, Chin-Hsiung, and 王慶雄. "Solid phase microextraction combined with ambient ionization mass spectrometry for high-throughput pharmacokinetic analysis of human plasma." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/57j7pq.
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博士國立中山大學
化學系研究所
107
The content of this thesis mainly includes the development of solid phase microextraction combined with ambient mass spectrometry for high-throughput analysis of drug for pharmacokinetic studies. Traditionally, liquid or gas chromatography tandem mass spectrometry requires lengthy and laborious extraction and concentration process so that it is prone to misconducts and experimental errors. Therefore a simple and high-throughput analytical approach is deemed necessary. In this thesis, the solid phase microextraction technique combined thermal desorption electrospray ionization mass spectrometry (SPME-TD-ESI/MS) approach is used to rapidly determine the concentration of drugs in pharmacokinetics samples. Polar, relatively non-polar, and combine of both adsorption materials SPME fiber were explored for direct immersion solid phase microextraction thermal desorption electrospray ionization mass spectrometry approach. In addition, a new developed in-tip SPME device was firstly disclosed and proved to use 1 μL plasma sample for each analysis. Experimental results shown that the average errors were less than 20% compared to LC-MS/MS method. Lower limit of quantification as low as 0.2 ng mL-1 and linear regression coefficient (r) values were above 0.995. The SPME extraction and instrument detection time was as short as 2.5 min. In overall, experimental results show that SPME-TD-ESI/MS approach is not only suitable for high-throughput analysis of pharmacokinetic samples. Polar and non-polar SPME fiber could also be flexibly combined for simultaneously extracting analytes of different polarity. The new design of in-tip SPME pushes down the sample volume to 1 μL plasma for each analysis greatly enhances its applicability in pharmacokinetics study especially in small animal models.
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Wall, Mark James. "Towards the Development of a Proteomics Workflow for High-throughput Protein Biomarker Discovery." 2010. http://hdl.handle.net/10222/12840.
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Two popular workflows exist for quantitative proteome analysis: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), with staining to visualize proteins, and multidimensional solution phase separations of isotopically labelled peptides coupled to mass spectrometry (MS). However, the development of an alternative strategy, which combines easy-to-read differential profiling as seen in 2D-PAGE, with the sensitivity of MS for detection and identification, is needed. This thesis presents work towards the development of a workflow for high-throughput protein biomarker discovery.
Multidimensional separations are vital to obtain sufficient fractionation of complex proteome mixtures. As a first dimension of separation, ion exchange chromatography (IEC) is a common choice, though it has yet to be thoroughly evaluated in terms of its effectiveness as a proteome prefractionation tool. This study used a defined set of protein standards to establish the resolution and proteome yield obtained through IEC. The evaluation uncovered significant bias in terms of protein recovery and separation.
To improve throughput of a multidimensional separation strategy, a multiplexed (8-column) reversed phase liquid chromatography (RPLC) platform was constructed. The system design allowed for even distribution of flow across all columns with limited cross-loading during sample loading. This system was directly coupled to matrix-assisted laser desorption/ionization (MALDI) through a novel well plate device. The Teflon wells allowed for high recovery and no cross-contamination during collection/spotting, improved throughput, and greatly reduced the number of sample manipulation steps.
An evaluation of MALDI MS, using the ThermoFisher vMALDI LTQ, for quantitative profiling was performed, employing the multiplexed LC-MALDI platform. The use of MALDI MS allowed for fast (< 5.5 hours) acquisition of quantitative data from isotopically differentiated samples partitioned over 640 fractions from two-dimensional LC. Proteins comprising 0.1% of the proteome were detected and quantified using this method.
Finally, the effects of varying concentrations of acetonitrile (ACN) upon the products generated from tryptic digestions were explored. Poor enzymatic efficiency in 80% ACN was found to be responsible for an increased concentration of peptides containing missed cleavage sites. These peptides often contained unique amino acid sequences, which were not detected from complete digestions, resulting in improved protein sequence coverage following MS analysis.
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Zhou, Ao. "Characterizing alternative splicing and long non-coding RNA with high-throughput sequencing technology." Diss., 2018. http://hdl.handle.net/1805/17771.
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Indiana University-Purdue University Indianapolis (IUPUI)Several experimental methods has been developed for the study of the central dogma since late 20th century. Protein mass spectrometry and next generation sequencing (including DNA-Seq and RNA-Seq) forms a triangle of experimental methods, corresponding to the three vertices of the central dogma, i.e., DNA, RNA and protein. Numerous RNA sequencing and protein mass spectrometry experiments has been carried out in attempt to understand how the expression change of known genes affect biological functions in various of organisms, however, it has been once overlooked that the result data of these experiments are in fact holograms which also reveals other delicate biological mechanisms, such as RNA splicing and the expression of long non-coding RNAs. In this dissertation, we carried out five studies based on high-throughput sequencing data, in an attempt to understand how RNA splicing and differential expression of long non-coding RNAs is associated biological functions. In the first two studies, we identified and characterized 197 stimulant induced and 477 developmentally regulated alternative splicing events from RNA sequencing data. In the third study, we introduced a method for identifying novel alternative splicing events that were never documented. In the fourth study, we introduced a method for identifying known and novel RNA splicing junctions from protein mass spectrometry data. In the fifth study, we introduced a method for identifying long non-coding RNAs from poly-A selected RNA sequencing data. Taking advantage of these methods, we turned RNA sequencing and protein mass spectrometry data into an information gold mine of splicing and long non-coding RNA activities.
2019-05-06
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(6618998), Zinia Jaman. "HIGH THROUGHPUT EXPERIMENTATION AS A GUIDE TO THE CONTINUOUS FLOW SYNTHESIS OF ACTIVE PHARMACEUTICAL INGREDIENTS." Thesis, 2020.
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Continuous flow chemistry for organic synthesis is an emerging technique in academia and industry because of its exceptional heat and mass transfer ability and, in turn, higher productivity in smaller reactor volumes. Preparative electrospray (ES) is a technique that exploits reactions in charged microdroplets that seeks to accelerate chemical synthesis. In Chapter 2, the flow synthesis of atropine, a drug which is included in the WHO list of essential of medicines and currently in shortage according to the U.S Food and Drug Administration (FDA)is reported.The two steps of atropine synthesis were initially optimized separately and then continuously synthesized using two microfluidic chips under individually optimized condition.The telescoped continuous-flow microfluidics experiment gave a 55% conversion with an average of 34% yield in 8 min residence time. In Chapter 3, a robotic HTE technique to execute reactions in 96-well arrays was coupled with fast MS analysis. Palladium-catalyzed Suzuki-Miyaura (S-M) cross-coupling reactions were screened in this system and a heat map was generated to identify the best reaction condition for downstream scale up in continuous flow.
In Chapter 4, an inexpensive and rapid synthesis of an old anticancer drug, lomustine,was synthesized. Using only four inexpensive commercially available starting materials and a total residence time of 9 min, lomustine was prepared via a linear sequence of two chemical reactions performed separately in two telescoped flow reactors. Sequential offline extraction and filtration resulted in 63% overall yield of pure lomustine at a production rate of 110 mg/h. The primary advantage of this approach lies in the rapid manufacture of lomustine with two telescoped steps to avoid isolation and purification of a labile intermediate, thereby decreasing the production cost significantly. A high throughput reaction screening approach based on desorption electrospray ionization mass spectrometry (DESI-MS) is described in Chapter 4 and 5 for finding the heat-map from a set of reaction conditions. DESI-MS is used to quickly explore a large number of reaction conditions and guide the efficient translation of optimized conditions to continuous flow synthesis that potentially accelerate the process of reaction optimization and discovery. Chapter 5 described HTE ofSNAr reactions using DESI-MS and bulk techniques with 1536 unique reaction conditions explored using both in DESI-MS and bulk reactors. The hotspots from the HTE screening effort were validated using a microfluidic system that confirmed the conditions as true positives or true.
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Hopper, Erin D. "Development and Application of a Mass Spectrometry-Based Assay for the High Throughput Analysis of Protein-Ligand Binding." Diss., 2009. http://hdl.handle.net/10161/1117.
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Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.
This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.
The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.
To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.
Dissertation
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(10971108), Yangjie Li. "REACTION ACCELERATION AT INTERFACES STUDIED BY MASS SPECTROMETRY." Thesis, 2021.
Find full textAbstract:
Various organic reactions, including important synthetic reactions involving C–C, C–N, and C–O bond formation as well as reactions of biomolecules, are known to be accelerated when the reagents are present in confined volumes such as sprayed or levitated microdroplets or thin films. This phenomenon of reaction acceleration and the key role of interfaces played in it are of intrinsic interest and potentially of practical value as a simple, rapid method of performing small-scale synthesis. This dissertation has three focusing subtopics in the field of reaction acceleration: (1) application of reaction acceleration in levitated droplets and mass spectrometry to accelerate the reaction-analysis workflow of forced degradation of pharmaceuticals at small scale; (2) fundamental understanding of mechanisms of accelerated reactions at air/solution interfaces; (3) discovery the use of glass particles as a `green' heterogeneous catalysts in solutions and systematical study of solid(glass)/solution interfacial reaction acceleration as a superbase for synthesis and degradation using high-throughput screening.
Reaction acceleration in confined volumes could enhance analytical methods in industrial chemistry. Forced degradation is critical to probe the stabilities and chemical reactivities of therapeutics. Typically performed in bulk followed by LC-MS analysis, this traditional workflow of reaction/analysis sequence usually requires several days to form and measure desirable amount of degradants. I developed a new method to study chemical degradation in a shorter time frame in order to speed up both drug discovery and the drug development process. Using the Leidenfrost effect, I was able to study, over the course of seconds, degradation in levitated microdroplets over a metal dice. This two-minute reaction/analysis workflow allows major degradation pathways of both small molecules and therapeutic peptides to be studied. The reactions studied include deamidation, disulfide bond cleavage, ether cleavage, dehydration, hydrolysis, and oxidation. The method uses microdroplets as nano-reactors and only require a minimal amount of therapeutics per stress condition and the desirable amount of degradant can be readily generated in seconds by adjusting the droplet levitation time, which is highly advantageous both in the discovery and development phase. Built on my research, microdroplets can potentially be applied in therapeutics discovery and development to rapidly screen stability of therapeutics and to screen the effects of excipients in enhancing formulation stabilities.
My research also advanced the fundamental understanding of reaction acceleration by disentangles the factors controlling reaction rates in microdroplet reactions using constant-volume levitated droplets and Katritzky transamination as a model. The large surface-to-volume ratios of these systems results in a major contribution from reactions at the air/solution interface where reaction rates are increased. Systems with higher surface-active reactants are subject to greater acceleration, particularly at lower concentrations and higher surface-to-volume ratios. These results highlight the key role that air/solution air/solution interfaces play in Katritzky reaction acceleration. They are also consistent with the view that reaction increased rate constant is at least in part due to limited solvation of reagents at the interface.
While reaction acceleration at air/solution interfaces has been well known in microdroplets, reaction acceleration at solid/solution interfaces appears to be a new phenomenon. The Katritzky reaction in bulk solution at room temperature is accelerated significantly by the surface of a glass container compared to a plastic container. Remarkably, the reaction rate is increased by more than two orders of magnitude upon the addition of glass particles with the rate increasing linearly with increasing amounts of glass. A similar phenomenon is observed when glass particles are added to levitated droplets, where large acceleration factors are seen. Evidence shows that glass acts as a ‘green’ heterogeneous catalyst: it participates as a base in the deprotonation step and is recovered unchanged from the reaction mixture.
Subsequent to this study, we have systematically explored the solid/solution interfacial acceleration phenomena using our latest generation of a high-throughput screening system which is capable of screening thousands of organic reactions in a single day. Using desorption electrospray ionization mass spectrometry (DESI-MS) for automated analysis, we have found that glass promotes not only organic reactions without organic catalysts but also reactions of biomolecules without enzymes. Such reactions include Knoevenagel condensation, imine formation, elimination of hydrogen halide, ester hydrolysis and/or transesterification of acetylcholine and phospholipids, as well as oxidation of glutathione. Glass has been used as a general `green' and powerful heterogeneous catalyst.
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Huang, Jing-Yi, and 黃靜怡. "Application of Liquid Chromatography-Tandem Mass Spectrometry for High Throughput Screening of Sedative-Hypnotics and Abused Drugs in Urine." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/dcga4k.
Full textAbstract:
碩士國立中興大學
化學系所
99
The people who has insomnia is increasing rapidly because of working pressure and fast pace of life. Prescription drugs used specifically for improving sleeping are called sedative hypnotics. These drugs include benzodiazepines and non-benzodiazepines such as zolpidem. People who use drugs for pain relief may become dependent and drug abuse can lead to drug dependence or addiction. Ketamine, like benzodiazepines, is a central nervous system depressant. It is primarily used for the induction and maintenance of general anesthesia, usually in combination with a sedative, and is a quick acting drug used in human and veterinary medicine. Ketamine has over the past few years been thought of as a “club drug”, and often used with sedative hypnotics. These drugs can cause dream-like states and hallucinations. Since drug abuse that alters human behavior and leads to crimes has become a serious problem throughout the world, it is important to develop a fast and accurate method to determine abused drugs. The aim of this study is to develop a rapid micro solid phase extraction (μSPE) combined with liquid chromatography/ tandem mass spectrometry (LC/MS/MS) for simultaneous determination 14 drugs of the third and fourth controlled drugs and metabolites in urine. The 14 target analytes are flunitrazepam, nimetazepam, triazolam, diazepam, estazolam, alprazolam midazolam, lorazepam, zaleplon, zopiclone, zolpidem, ketamine and two metabolits of ketamine. The highly selective reaction monitoring (H-SRM) scan mode was used in this study to improve selectivity and sensitivity. The linearity ranges were between 0.5～500 ng/mL with coefficients of determination (R2) above 0.9916 for 14 drugs. The limit of detection (LOD) ranged from 0.15～6 ng/mL ng/mL. The average recoveries at 10 ng/mL were 78.0～103 % and the relative standard deviations (RSD％) were 7.0～18.2 %, precisions of interday and intraday were 3.7～15.1 % and 5.6～17.9 % respectively. Thus, the established method should be useful in determination of abused drugs in real urine samples.
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Maděra, Milan. "Development of Methods for High-Throughput Enrichment of Glycoproteins and Glycopeptides Employing Multiple Lectin Affinity Chromatography/Tandem Mass Spectrometry." Doctoral thesis, 2006. http://www.nusl.cz/ntk/nusl-374322.
Full textAbstract:
5 CONCLUSIONS This thesis surrrnrarizes the development of a multimethodological analýical approach employing microcolumn lectin affi1t1,9fuom1tography coupled to highir".otu,to; separation and dete9tion techniques, in order to facilitate the enrichmení of glyco-proteins aná giycopeptioes originating from small quantities of real samples. This is u"toauy a sipificant-ítep towara satis$'ing the demands of contemporary glycoproteomics, which rely orirurt *a small scale analyses, allowing the identification of low abundant protein components' The overď conclusions and contribution to the contemporaÍy scierrce are drawn in tbree followine chaoters. 5.1 combining Lectin Microcolumns with High-Resolution separation Techniques for Enrichment of Glycoproteins and Glycopeptides This section describes coupling small-scďe lectin aÍfinity chromatograpby on-line to high- resolution separation and detection techniques, with the utiliý of a microcolumn loaded with a lectin immobilized onto macroporous silica. Experimental results, involving optimization of coupling procedure, fabrication of lectin microcolumns, verification of tteir Uin&ng efficiency and their possibility of interfacing on-line with nano LC-MS/]víS, me summarizžd into thé following conclusions; o optimized coupling procedure involved resuspending only 125...
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Spiegel, Christopher. "Development of a high-throughput shotgun-mass spectrometry method for qualitative and quantitative analysis of major mammalian brain gangliosides." 2019. https://tud.qucosa.de/id/qucosa%3A72379.
Full textAbstract:
The goal of this thesis was to develop a high-throughput shotgun-MS lipidomics method to qualitatively and quantitively analyze the major mammalian brain ganglioside classes: GM1, GD1, GT1 and GQ1. As a starting point for the method to be developed, a modified ganglioside extraction method from Svennerholm and Ladisch was used (Svennerholm and Fredman, 1980; Ladisch and Gillard, 1985). The efficiencies and the impact of different extraction procedures to the overall performance were evaluated with a software called OptiVal™. The evaluation showed that the most important steps of the protocol are the salt concentration of the water phase during the 2-phase extraction, and 10 mM NaCl yielded the best sensitivity. Also, the number of washing steps with water during reverse solid phase extraction using C18 resin has a significant effect. The next step was to find suitable standards for quantification of the individual ganglioside classes. Since deuterated and alike ganglioside standards were commercially not available, we initially used a deuterated PE standard with limited success. A collaboration with the Ludger Johannes lab provided us with modified C17-ganglioside standards. The term “modified” describes the enzymatic exchange of the fatty acid in the hydrophobic tail by a 17-carbon atom long fatty acid. Since odd numbered fatty acids occur very rarely in nature, it is possible to use the measured intensity of the modified ceramide headgroup of 35:1 (Sphingosine C18:1 + Fatty Acid C17:0) to quantify natural gangliosides. Ideally, we would need to have a fitting modified C17-ganglioside standard for each class to be quantified. Since first only GM1 as a modified standard was available, it was necessary to determine response factors (RFs) for the ganglioside classes GD1, GT1 and GQ1. RFs were assessed empirically by titrating a variety of equimolar concentrations of the modified C17-GM1 standard versus wildtype standards of the other ganglioside classes. After establishment of the RFs it was possible to determine the limits of detection (LOD) and quantification (LOQ) for the ganglioside classes GD1, GT1 and GQ1 - with regard to the modified C17-GM1 standard. When the modified C17-standards for GD1 and GT1 became available, I was able to find out whether the correct internal standards are superior to the proxy method via response factors. The results clearly showed that the use of a correct class standards is preferable. For GQ1 no modified C17-standard was obtainable, therefore this class still has to be quantified via RFs. Experiments showed that the modified C17-GT1 standard is best suited for that. Another major goal was to integrate the ganglioside method into the general lipid analysis workflow of the high-throughput shotgun mass spectrometry platform that we were using. To achieve these goals adjustments on the evaluated (=old) protocol had to be done. These adjustments included changes in the extraction steps from the Svennerholm & Ladisch more into the direction of a Bligh & Dyer based extraction method. This meant abandoning the 2-phase extraction step as well as the chloroform/methanol/water (C/M/W) 4:8:3 extraction, in favor of a C/M 10:1 followed by a C/M 2:1 extraction of 150 mM ammonium-bicarbonate water solution. The goal behind this was to enable a combination of the global lipidome extraction (Surma et al., 2015) with the ganglioside extraction. Another important improvement was scaling up the extraction process. The use of standard single solid phase extraction (SPE) cartridges was limiting the extraction throughput to only 24 samples at a time, therefore the single SPE cartridges were replaced with the 96-well SPE SOLA™ plates. To process the SOLA™ plates it was necessary to establish the usage of a vacuum manifold. Combined, these changes lowered the overall process time of the protocol from nearly two working days to one working day, without significant loss of sensitivity regarding the measured sample concentrations. This was assessed by performing the mouse brain tissue titration experiment, with all three modified C17-ganglioside class standards GM1, GD1 and GT1. Finally, the established method was applied to investigate the difference in ganglioside levels in the cerebellum compared to the brain hemispheres in mice of different age. First the C/M 10:1 and 2:1 extraction was done for the analysis of all non-ganglioside lipids in the sample. The leftover water phase was then loaded onto the SOLA™ plates and processed with the new protocol. The results matched the given goals - to establish a protocol to measure and quantify the four major brain ganglioside classes in combination with the global lipidomics in a high-throughput manner - and thus were a success. To the best of our knowledge, this was the first time such a broad lipidomic measurement has been performed, hence no other studies exist to which the outcome could be compared.
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Mirnaghi, Fatemeh Sadat. "High-throughput analysis of biological fluids using 96-blade (thin-film) solid phase microextraction system." Thesis, 2012. http://hdl.handle.net/10012/7175.
Full textAbstract:
The initial research of this thesis involves the evaluation of different strategies for developing diverse chemistries of highly stable coatings for the automated 96-blade (thin-film) solid phase microextraction (SPME) system. Thin-film geometry increases the volume of extractive phase, and consequently improves the sensitivity of the analysis. Sol-gel technology was used for the preparation of octadecyl (C18)-silica gel thin-film coating. The evaluation of the C18-silica gel SPME extractive phase resulted in stable physical and chemical characteristics and long-term reusability with a high degree of reproducibility.
Biocompatible polyacrylonitrile (PAN) polymer was used for the preparation of particle-based extractive phases in order to improve the biocompatible characteristics of SPME coatings for the extraction from biological samples.
Three different immobilization strategies were evaluated for developing highly stable coatings for the automated 96-blade SPME system. The spraying was found to be the optimal method in terms of stability and reusability for long-term use.
The optimized C18-PAN coating demonstrated improved biocompatibility, stability, and reusability for the extraction of benzodiazepines from human plasma in comparison with those of C18-silica gel coating.
To improve the biocompatible properties of the C18-PAN SPME coating for long-term direct analysis from whole blood, different modification strategies were studied and evaluated. The modification of the coating with an extra layer of biocompatible polyacrylonitrile resulted in significant improvement in the blood compatibility in long-term use.
‘Extracted blood spot’ (EBS) sampling was introduced as a novel approach to overcome the limitations of dried blood spot sampling. EBS includes the application of a biocompatible SPME coating for spot sampling of blood or other biofluids. The compatibility of EBS sampling with different analytical methods was demonstrated. The utilization of EBS as a fast sampling and sample preparation method resulted in a significant reduction of matrix effects through efficient sample clean-up.
Modified polystyrene-divinylbenzene (PS-DVB)-PAN and phenylboronic acid (PBA)-PAN 96-blade SPME coatings were developed and evaluated for the extraction of analytes in a wide range of polarity. These coatings demonstrated efficient extraction recovery for both polar and non-polar groups of compounds, and presented chemical and mechanical stabilities and reproducible extraction efficiencies for more than 100 usages in biological sample.
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"High-throughput quantitative profiling of serum N-glycome by MALDI-TOF mass spectrometry and N-glycomic fingerprint of liver fibrosis." 2008. http://library.cuhk.edu.hk/record=b5893558.
Full textAbstract:
Kam, Kin Ting.Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 169-192).
Abstracts in English and Chinese.
Chapter 1. --- Abstract --- p.ii
English --- p.ii
Chinese --- p.v
Chapter 2. --- Acknowledgments --- p.vii
Chapter 3. --- Abbreviations and N-glycan representation --- p.viii
Chapter 4. --- Introduction --- p.1
Chapter 5. --- Review of Literatures --- p.2
Chapter 5.1. --- Introduction to Liver Fibrosis --- p.2
Chapter 5.1.1. --- Pathogenesis of Liver Fibrosis --- p.2
Chapter 5.1.2. --- Changes of liver architecture - basis of liver fibrosis diagnosis --- p.4
Chapter 5.2. --- Current Diagnosis of Liver Fibrosis - from Biopsy Examination to Serum Test --- p.5
Chapter 5.3. --- Glycomics and its Potential as Biomarkers --- p.9
Chapter 5.3.1. --- Overview of Biochemical and Functional Characteristics of Glycan --- p.13
Chapter 5.3.2. --- N-linked and O-linked Glycosylations - A Valuable Source of Biomarkers --- p.15
Chapter 5.3.3. --- Glycomics 一 An Uprising Approach for Biomarker Discovery --- p.17
Chapter 5.3.4. --- Human Proteome Organisation Human Disease Glycomics/Proteome Initiative --- p.19
Chapter 5.3.5. --- Recent Applications of Glycomics to Biomarker Discovery --- p.20
Chapter 5.4. --- Current Technologies for Glycomic Study --- p.22
Chapter 5.4.1. --- MALDI-TOF MS --- p.22
Chapter 5.4.2. --- Lectin Microarray --- p.25
Chapter 5.4.3. --- Liquid Chromatography --- p.27
Chapter 5.4.4. --- Capillary Electrophoresis --- p.29
Chapter 5.4.5. --- Quantitative Profiling of Tissue Glycome --- p.31
Chapter 6 --- Project Rationales and Objectives --- p.36
Chapter 7 --- Section 1: Methodology Development of Quantitative N- glycomic Profiling --- p.37
Chapter 1. --- Introduction --- p.37
Chapter 2. --- Method and Materials --- p.39
Chapter 3. --- Results --- p.46
Chapter 4. --- Discussion --- p.65
Chapter 5. --- Conclusion --- p.71
Chapter 8. --- Section 2: Serum N-glycomic Profile as Biomarker for Liver Fibrosis 一 Pilot Study --- p.73
Chapter 1. --- Introduction --- p.73
Chapter 2. --- Method and Materials --- p.75
Chapter 3. --- Results --- p.79
Chapter 4. --- Discussion --- p.86
Chapter 5. --- Conclusion --- p.94
Chapter 9. --- Section 3: Serum N-glycomic Profile as Biomarker for Liver Fibrosis -Verification Study --- p.96
Chapter 1. --- Introduction --- p.96
Chapter 2. --- Method and Materials --- p.98
Chapter 3. --- Results --- p.104
Chapter 4. --- Discussion --- p.137
Chapter 5. --- Conclusion --- p.152
Chapter 10. --- General Discussion --- p.153
Chapter 11. --- Conclusion --- p.167
Chapter 12. --- Original Data --- p.168
Chapter 13. --- References --- p.169
Chapter 14. --- Publications --- p.196
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Lin, Jing-yun, and 林靜芸. "The Development of High Throughput Screening Technique for Fumonisins by Matrix-Assisted Laser Desorption/Inoization Time-of-Flight Mass Spectrometry." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/58678809244737949069.
Full textAbstract:
碩士朝陽科技大學
應用化學系碩士班
99
Fumonisins are secondary metabolites of Fusarium moniliforme, mainly in corn and corn-based products. It may cause flu-like symptoms on pig and result in wrong treatment. Moreover, fumonisins are associated with human esophageal cancer. Fumonisin B1 (FB1) is the most toxic and most natural abundant type (70%) in Fumonisins. The official analysis method for FB1 is using disposable immunoaffinity column (NT$≧300) for extraction then following derivatization before HPLC-FLD measurement. However, the cost of the specific column and the time consuming are not good for high throughput analysis, especially for the non-contaminated samples. If there is a low cost and high throughput screening methods to filter out most uncontaminated samples, a lot of time consuming and cost would be avoided. Thus, we develop a low-cost, high sensitivity screening method for FB1 by MALDI-TOF MS to achieve this goal. In this method, the pretreatment just need solvent (MeOH/H2O) extraction following centrifugation, then detected by MALDI-TOF/MS using α-CHCA as the matrix and Cs+ to increase sensitivity to 1 ppb. The detection limit would be only 1 ppm without Cs+. The cost of sample preparation for each sample is less than NT$ 10.0 dollars and analysis time for each sample is only 1 min or less. This is in line with current environmental protection concept of green chemistry.
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Hu, Li-Wen, and 胡瓈文. "The Development of High Throughput Screening Technique for Citrinin by Matrix-Assisted Laser Desorption/Inoization Time-of -Flight Mass Spectrometry." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/73564751354971655339.
Full textAbstract:
碩士朝陽科技大學
應用化學系
102
Citrinin (Citrinin, CIT) , a kind of mycotoxins with teratogenic toxicity and harmful to kidney and liver, is a metabolite of Monascus fermentation during generating red pigment. Since more and more health-related Monascus fermentation products are introduced to the market, the risk of citrinin contaiminations may increase. Therefore , I develope a low-cost, and rapid screening method to detect CIT by using matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI-TOF MS) . On this method, the pretreatment process requires only about 2 mL solvent to extract the sample centrifuged, the supernatant can be detected directly by MALDI-TOF/MS. When using α-CHCA as matrix, the detection limit is 1 ppm for standard, after adding α-cyclodextrin in the matrix, the limitation of detection can be improved 10 times to the 100 ppb. In the real specimen of wheat and red yeast pancake the detection limit can reach to 200 ppb ; for red yeast rice and red yeast capsules the detection limit can reach to 1 ppm. This method requires very low cost (NT ≦ 10) for each sample and the detection time is less than one minute. By this method, I may avoid unnecessary waste of materials and time consuming. This is in line with current environmental protection concept of green chemistry.
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Lin, Mei-wun, and 林美文. "The Development of High Throughput Screening Technique for Deoxynivalenol by Matrix-Assisted Laser Desorption/Inoization Time-of -Flight Mass Spectrometry." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/45326567672230099451.
Full textAbstract:
碩士朝陽科技大學
應用化學系碩士班
97
Deoxynivalenol (DON or vomitoxin) is a mycotoxin and is one of the most common and abundant naturally occurring trichothecenes found in corn, wheat, and barley. Conventional analysis methods in the sample preparation and detection process is cost and time consuming, and therefore not suitable for high throughput screening. In Taiwan, the official analysis method for Deoxynivalenol is using immunoaffinity column coupling with high performance liquid chromatography (HPLC) which has the detection limit of 60 ppb. The cost of the specific column and the time consuming are not good for high throughput analysis. If there is a low cost and high throughput screening methods to filter out most uncontaminated samples, a lot of time consuming and cost would be avoided. Here we report a low cost and high throughput screening method for the analysis of Deoxynivalenol using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). First, This method includes two part, sample preparation method based on celite is simple and low cost. Second, high throughput of the MALDI-TOF MS supply a detection. An ionic liquid matrix (Et3N-a-CHCA) is used to obtain low matrix noise mass spectra for low molecular weight Deoxynivalenol and the Deoxynivalenol signal was enhanced by adding KCl via K+ cationization. The method of sample preparation cost for each sample is NT$ 10.0 dollars, and a sample analysis time is only 1 min, detection limit can reach to 10 ppb. Key words：deoxynivalenol, MALDI-TOF MS
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Hsieh, Cheng-Chuan, and 謝政娟. "High-Throughput Chip-Based Solid Phase Extraction Coupling with Inductively Coupled Plasma-Mass Spectrometry for Online Determination of Trace Elements." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/30122884080524470557.
Full textAbstract:
碩士國立清華大學
生醫工程與環境科學系
99
Although inductively coupled plasma-mass spectrometry (ICP-MS) is a very powerful technique for trace metal analysis, the sample concomitants often lead to significant spectral interference and/or loss of sensitivity during the determination process. Therefore, incorporating an efficient online pretreatment technique to ICP-MS is considered as an indispensable alternative to concentrate analyte ions and to minimize the adverse effects caused by the concomitant matrices. Among various sample pretreatment techniques, solid-phase extraction (SPE) method is generally superior in terms of their simplicity and efficacy. Over the past decade, several chip-based SPE techniques have been extensively applied to analytical works. Its unique characteristics of high surface-to-volume ratio and short molecular diffusion distance enable a new pretreatment paradigm that has the potential to effectively separate analyte species from sample matrix. Even so, the realization of high-throughput analyses for chip-based SPE techniques remains difficult and rare until now. In general, increasing the operation flow rate is straightforward for achieving high-throughput SPE procedures. However, microdevices used for high-throughput analyses are often limited to large increase in the hydrodynamic resistance with increasing operation flow rate. Also, complete mixing of two fluids (sample and buffer solutions) within a reasonable time is quite difficult due to the low Reynolds number on the micro scale. To overcome the limitations, in this study, a polytetrafluoroethylene (PTFE) bead was implanted into the mixing chamber to change the streamline of solutions for efficient mixing and then the chip-based SPE device which composed of circularly multichannel pattern was employed to carry out the SPE procedures. Based on our results, this hyphenated system could effectively eliminate the salt-interference resulted from the salt matrices and preconcentrate desired elements even the extraction flow rate was extremely high. In addition, dilute samples were further used to demonstrate the first successfully high-throughput on-chip SPE of trace metal ions from high-salt content samples.