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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Metabolômica como ferramenta em taxonomia: O modelo em Arnica. Metabolomics in plant taxonomy: The Arnica model / Metabolomics in plant taxonomy: The Arnica model

Madeleine Ernst 23 August 2013 (has links)
Taxonomia vegetal é a ciência que trata da descrição, identificação, nomenclatura e classificação de plantas. O desenvolvimento de novas técnicas que podem ser aplicadas nesta área de conhecimento é essencial para dar suporte às decisões relacionadas a conservação de hotspots de biodiversidade. Nesta dissertação de mestrado foi desenvolvido um protocolo de metabolic fingerprinting utilizando MALDI-MS (matrix-assisted laser desorption/ionisation mass spectrometry) e subsequente análise multivariada utilizando scripts desenvolvidos para o pacote estatístico R. Foram classificadas, com base nos seus metabólitos detectados, 24 plantas de diferentes famílias vegetais, sendo todas elas coletadas em áreas da Savana Brasileira (Cerrado), que foi considerada um hotspot de biodiversidade. Metabolic fingerprinting compreende uma parte da Metabolômica, i.e., a ciência que objetiva analisar todos os metabólitos de um dado sistema (celula, tecído ou organismo) em uma dada condição. Comparada com outros métodos de estudo do metaboloma MALDI-MS apresenta a vantagem do rápido tempo de análise. A complexidade e importância da correta classificação taxonômica é ilustrada no exemplo do gênero Lychnophora, o qual teve diversas espécies incluídas neste estudo. No Brasil espécies deste gênero são popularmente conhecidas como \"arnica da serra\" ou \"falsa arnica\". Os resultados obtidos apontam similaridades entre a classificação proposta e a classificação taxonômica atual. No entanto ainda existe um longo caminho para que a técnica de metabolic fingerprinting possa ser utilizada como um procedimento padrão em taxonomia. Foram estudados e discutidos diversos fatores que afetaram os resultados como o preparo da amostra, as condições de análise por MALDI-MS e a análise de dados, os quais podem guiar futuros estudos nesta área de pesquisa. / Plant taxonomy is the science of description, identification, nomenclature and classification of plants. The development of new techniques that can be applied in this field of research are essential in order to assist informed and efficient decision-making about conservation of biodiversity hotspots. In this master\'s thesis a protocol for metabolic fingerprinting by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) with subsequent multivariate data analysis by in-house algorithms in the R environment for the classification of 24 plant species from closely as well as from distantly related families and tribes was developed. Metabolic fingerprinting forms part of metabolomics, a research field, which aims to analyse all metabolites, i.e., the metabolome in a given system (cell, tissue, or organism) under a given set of conditions. Compared to other metabolomics techniques MALDI-MS shows potential advantages, mainly due to its rapid data acquisition. All analysed species were collected in areas of the Brazilian Savanna (Cerrado), which was classified as \"hotspot for conservation priority\". The complexity and importance of correct taxonomic classification is illustrated on the example of the genus Lychnophora, of which several species also have been included into analysis. In Brazil species of this genus are popularly known as \"arnica da serra\" or \"falsa arnica\". Similarities to taxonomic classification could be obtained by the proposed protocol and data analysis. However there is still a long way to go in making metabolic fingerprinting by MALDI-MS a standard procedure in taxonomic research. Several difficulties that are inherent to sample preparation, analysis of plant\'s metabolomes by MALDI-MS as well as data analysis are highlighted in this study and might serve as a basis for further research.
22

Performance of SpheriCal® standards as calibrants for IgG glycopeptide analysis using MALDI-MS / Användning av SpheriCal®-standarder som kalibranter för analys av IgG glykopeptider med MALDI-MS

Bubic, Sandra, Kjellberg, Martin, Samuelsson, Ludvig January 2022 (has links)
I dagens samhälle finns alla möjliga sorters sjukdomar - alltifrån den vanliga säsongsinfluensan till mer allvarliga infektioner och kroniska sjukdomar som cancer och Alzheimers. Därför finns även ett stort intresse för att kunna ställa rätt diagnos samt söka möjliga behandlingar och att bota dessa sjukdomar. Ett sätt att göra detta på är genom att använda biomarkörer, IgG (Immunoglobulin G) är en biomarkör som visat sig vara passande i detta syfte. Vid användning av MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionization - Time of Flight - Mass Spectrometry) krävs dock anrikning av glykopeptiderna för att exakta resultat ska erhållas. Genom att utnyttja de starka hydrofila interaktionerna mellan glykanerna, som inte finns hos icke-glykopeptider, kan de glykosylerade peptiderna bli anrikade för att högre intensitet ska erhållas i spektrat. Därav, är syftet med detta kandidatexamensarbete att undersöka huruvida SpheriCals® kalibreringsstandarder är passande i syftet att möjliggöra- samt förbättra användandet av biomarkörer i diagnostik och andra medicinska appliceringar där prov analyseras med MALDI-TOF-MS. Hittills har andra peptidbaserade kalibranter använts och det är därför önskvärt att jämföra dessa med SpheriCal® för att se om den sistnämnda genererar mer exakta mätningar. Det första steget var glykosylering för att få glykopeptider från IgG. Därefter genomfördes experiment med både interna och externa kalibreringsmetoder för naturliga, renade samt till viss del nedbrutna peptider och mänskliga prover från friska individer samt från patienter med COVID-19. Dessa experiment genomfördes i olika matriser, mer exakt i DHB (2,4-dihydroxibensoesyra) och HCCA (α-cyano-4-hydroxikanelsyra) En sammanfattning av resultaten visar att SpheriCal®-kalibranter möjliggör mätningar med hög noggrannhet och små fel för uppmätt m/z (massa mot laddning) för både intern och extern kalibrering vid analys av IgG glykopeptider. Vid extern kalibrering gav SpheriCal®-APH mätningar med väsentligt högre exakthet än för kalibrering med mer traditionellt använt PCS i både HCCA och DHB. / Our world is full of all different kinds of diseases - everything from the regular flu to more severe infections and chronic illnesses such as cancer and Alzheimer's disease. It is therefore of interest to be able to establish a diagnosis, and thus search possible treatments and cures. One way to do this is by using biomarkers and IgG (Immunoglobulin G) has shown to be suited as one. When using MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionization -Time of Flight - Mass Spectrometry), enrichment of the glycopeptides is required to provide an accurate analysis. Hence, by utilizing the strong hydrophilic interactions of glycans, which do not exist in non-glycopeptides, the glycosylated peptides can be enriched to achieve higher intensity in the spectra. That is why the aim of this bachelor’s degree project is to investigate if SpheriCal® calibrant standards are appropriate for the purpose of enabling and bettering the use of biomarkers in diagnostics and other medical areas when analyzing samples with MALDI-TOF-MS. Until now, other peptide-based calibrants have been used. Therefore, it has been desirable to compare the two to showcase whether SpheriCal® generates more accurate measurements. An initial step was glycosylation in order to obtain glycopeptides of IgG. Following that, tests were carried out with both internal- as well as external calibration methods with natural, purified and partly digested peptides and human samples of healthy- and COVID-19 infected patients. Furthermore, different matrices were tested, more specifically, DHB (2,5-dihydroxybenzoic acid) and HCCA (α-Cyano-4-hydroxycinnamic acid). To conclude the results, they showed that SpheriCal® calibrants generate high accuracy with small m/z (mass to charge) errors, for both internal- as well as external calibration methods, when analyzing IgG glycopeptides. For external calibration, SpheriCal®-APH showed significantly higher mass accuracy than conventionally used PCS in both HCCA and DHB.
23

Le marquage des peptides avec des métaux et détection par MS et l'optimisation des procédures de l'extraction de métalloprotéin dans les échantillons biologiques à des fins de protéomique / Peptide labeling with metals using MS detection and optimization of metalloprotein extraction procedures in biological samples with proteomic purposes

Lyrio Tenorio Correia, Carolina 10 March 2014 (has links)
Ce travail a développé une nouvelle méthode pour l'identification et la quantification des peptides, par l'optimisation de certaines stratégies disponibles appropriées pour le marquage des peptides avec des métaux lanthanide, une séparation par nano-HPLC et détection UV, et suivi par MALDI MS. Tout d'abord, les peptides ont été marqués avec les trois métaux lanthanides différents et un réactif fonctionnel - DOTA. Les résultats montrent que la réaction de transformation en dérivé à l'aide du réactif chélateur DOTA-NHS-ester a été efficace pour des peptides individuels et des mélanges de peptides, vérifiées à partir de la relation m/z obtenue par MALDI MS. L'application optimisée d’un complexe (Cytochrome C digest) a montré des résultats comparables à ceux obtenus avec des peptides modèles. En parallèle, nous avons effectué l’optimisation pour la purification de métalloprotéine dans la bile de poisson, qui est signalée entant que biomarqueurs de contamination métallique de l'environnement. Des procédures différentes (différents moments de centrifugation et différentes températures de traitement thermique) et les agents (DTT, β-mercaptoéthanol et TCEP) réduisant ont été apliqués pour purifier les MT isolées de la bile et du foie des poissons (Oreochromis niloticus). Des analyses spectrophotométriques ont été utilisées pour quantifier les échantillons de MT, et le gel SDS-PAGE a été utilisé pour évaluer qualitativement les différents résultats de la procédure. Chaque procédure a en suíte été évaluée statistiquement, une méhtode des surfaces de réponse a été appliquée. Les MT de la bile semblent être plus adéquate pour la surveillance de l'environnement en ce qui concerne l'exposition récente à des xénobiotiques qui peuvent influer sur l'expression protéomique et metalloproteomique de cette matrice biologique. Une procédure d’exposition à des métaux dans le laboratoire a montré que les métaux étaient significativement importante pour l’évaluation de la contamination à partir de la quantification de MT, selon le traitement de données par une techinique de réseau neural. / This work developed a new method for the identification and quantification of peptides, by optimizing some of the available strategies suitable for labeling peptides with lanthanide metals with subsequent separation by nano-HPLC with UV detection, matrix-assisted laser desorption ionization-mass spectrometry (MALDI MS). First, peptides were labeled with the three different lanthanide metals using a functional DOTA-based reagent. The results demonstrate that the derivatization reaction using the chelating reagent DOTA-NHS-ester was effective for single peptides and peptide mixtures, verified from the m/z relation obtained by MALDI MS. The application of the optimized method in a more complex matrix (Cytochrome C digest) showed results comparable to those obtained with model peptides. In parallel, environmental analyses were conducted, by performing the standardization of metalloprotein purification in fish bile, since this matrix has been reported as a biomarker for environmental metal contamination. Different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, β-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated. A response surface methodology was applied for bile samples, in order to further evaluate the responses for this matrix. In an environmental context, biliary MT was lower than liver MT, and, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix. A procedure for exposure to metals in the laboratory showed that some metals are significantly important for the assessment of contamination from the quantification of MT, according to the data processing by atifical neural network (ANN).
24

Développement du couplage électrophorèse capillaire-spectrométrie de masse à source MALDI : applications à la caractérisation de protéines / Development of coupling capillary electrophoresis - mass spectrometry MALDI source : applications to the characterization of proteins

Biacchi, Michael 23 September 2014 (has links)
Au cours de ce travail, nous avons mis au point une nouvelle interface CE/MALDI-MS automatisée, équipée d’une cellule UV/visible déportée, et d’une distribution automatique de matrice intégrée. Ce nouveau système a été évalué sur des mélanges différents de protéines entières, de digestats de protéines et d’anticorps monoclonaux (mAbs). Les résultats obtenus lors de cette évaluation ont montré la complémentarité de la nouvelle interface avec les systèmes analytiques classiques. De plus, nous avons montré la première séparation et analyse de mAbs entier par CE/MALDI-MS. Dans un second travail, la nouvelle interface a été utilisée pour effectuer la première analyse Top Down de proteine entière et de mAbs par collecte et enrichissement de fraction. Cette stratégie a montré la répétabilité du système permettant de séparer des analytes d’intérêt et d’enrichir les dépôts MALDI jusqu'à de très hautes quantités d’analytes permettant l’obtention de spectre Top Down. Au cours du troisième travail, le nouveau système CE/MALDI-MS a été utilisé dans une stratégie originale à 2 dimensions de séparation et de collecte d’isoformes de mAbs entiers ou partiellement digérés suivi d’infusions et analyses à l’aide du nanosprayer CESI. Pour cela, nous avons mis au point des conditions électrophorétiques dit « asymétriques » séparant les mAbs dans des conditions très salées mais collectés dans un milieu compatible avec l’ESI-MS. Cette stratégie inédite a permis d’effectuer la première séparation et caractérisation de mAbs par CE-MS. Parallèlement, nous avons mis au point le premier dosage plasmatique d’ITPP par MALDI-TOF MS et surtout la création de CEToolbox, une application Androïd gratuite pour smartphone et tablette permettant le calcul des principales grandeurs mathématiques pour la caractérisation et l’optimisation des séparations par électrophorèse capillaire. / In this work, we developed a new interface CE/MALDI-MS automated, equipped with a UV/visible cell remote, and integrated automatic distribution of matrix. This new system has been evaluated on different mixtures of intact protein, digested protein and monoclonal antibodies (mAbs). The results obtained during this evaluation showed the complementarity of the new interface with conventional analytical systems. Furthermore, we have shown the first separation and analysis of mAbs by CE/MALDI-MS. In a second work, the new interface was used to perform the first Top Down analysis for intact protein and mAbs by fraction collection and enrichment. This strategy has shown the repeatability of the system for separating analytes and the enrichissment the MALDI deposits up to very high amounts compatible for Top Down approach. In the third work, the new system CE/MALDI-MS has been used in an original 2-dimensional strategy of separating and collecting intact mAbs isoforms or partially digested followed by infusions and analyzes with CESI nanosprayer. For this, we have developed electrophoretic condition so-called "asymmetric" allowing the separation of mAbs under very salty conditions but collected in a totally compatible solution with ESI-MS. This novel strategy allowed for the first separation and characterization of mAbs by CE-MS. Meanwhile, we have developed the first plasma level of ITPP by MALDI-TOF-MS and particularly the creation of CEToolbox as a Free Android application for smartphone and tablet enabling the calculation of the main mathematical quantities for characterization and optimization of CE separations.
25

Nouvelles approches par spectrométrie de masse pour la caractérisation de systèmes archéologiques et biologiques : application à l'étude de cheveux de momies préhispaniques de la côte andine / New approaches by mass spectrometry for the characterization of archaeological and biological systems : application to the study of hair of prehispanic mummies from the Andean coast

Fresnais, Margaux 21 September 2016 (has links)
Les cheveux constituent un matériau de choix en archéométrie pour l’étude des civilisations anciennes. L’étude moléculaire de cheveux de momies et de leur protéome peut apporter de précieuses informations sur la composition, la préservation et l’environnement de la fibre. Une approche protéomique bottom-up dédiée à l’analyse en MS des protéines de cheveux de momies a été donc implémentée. Celle-ci a permis l’identification des protéines capillaires de cheveux anciens à partir de quantités minimales, ainsi que la caractérisation de leur état de conservation. Cette approche a été associée à une stratégie interdisciplinaire, intégrant également des analyses structurelles par FTIR, et élémentaires par SEM-EDS, XRF et PIXE. Enfin, le couplage direct TLC-MALDI-MS a été mis en place pour la caractérisation de systèmes biologiques et archéologiques, complexes et précieux. / Hair is an ideal material for the archaeometric study of past civilizations. Although it is rarely described, molecular study of hair and its proteome can provide precious clues on the ancient hair composition, its preservation state, as well as its environment. Here, we describe the implementation of a bottom-up proteomic approach for the mass spectrometry analysis of proteins from mummy hair. Through this approach, it was possible to identify the ancient hair proteins from a minimal initial amount of sample, and to characterize their molecular conservation state. This study was associated to an interdisciplinary project that also integrates structural analyses by FTIR and elemental analyses by SEM-EDS, XRF and PIXE. Finally, TLC-MALDI-MS hyphenation was implemented for the characterization of biological and archaeological systems, which are also complex and precious.
26

Massenspektrometrische Differenzierung bakterieller Infektionserreger mit dem Vitek MS-Routinetest-System (IVD) / Mass spectrimetric identification of bacteria by Vitek MS Routine lab System (IVD)

Menzer, Alexander January 2017 (has links) (PDF)
Untersucht wurde die Performance des Vitek MS-Systems anhand eines Keimspektrums von 1357 Isolaten im Zeitraum von Oktober 2011 bis April 2014. Das untersuchte Kollektiv bestand aus Isolaten der mikrobiologischen Routinediagnostik (n=1173), aus Stammsammlungen des Nationalen Referenzzentrum für Meningokokken und Haemophilus influenzae (n=128), sowie offizieller Stammsammlungen wie ATCC, DSMZ, LSM und anderen (n=56). Die Ergebnisse wurden entweder mit einer bereits vorhandenen Stammbezeichnung oder durch eine bzw. Kombinationen mehrerer molekularbiologischer (PCR der Teilabschnitte von Haushaltsgenen: 16S-rRNA-Untereinheit, sodA, recA) oder biochemischen Differenzierungsmethoden (Vitek 2, API-Systeme) verglichen. Mangels Referenzergebnisses wurden 25 Isolate (etwa 1,8%) aus der weiteren Betrachtung ausgeschlossen. Die verbliebenen 1332 Isolate wurden in 28 Bakterienordnungen, 109 Genera und 269 Spezies unterteilt. Auf Speziesebene konnten 1180 (etwa 88,6%) zum Vergleich herangezogen werden, da durch die Referenzmethoden nicht immer eine zuverlässige Speziesdifferenzierung gelang. Diese Referenzergebnisse wurden mit den Ergebnissen des Vitek MS-Systems verglichen. Eine Übereinstimmung zeigte sich auf Genusebene bei 86% (1157 von 1332 Isolaten) und auf Speziesebene bei 80% (944 von 1180 speziesdifferenzierten Keimen) der ausgewählten Stämme. Im Vergleich der Korrelationen der Vitek MS-Identifikationen und den Referenzergebnissen zeigte sich eine durchgehend gute Korrelation innerhalb der unterteilten Bakterienordnungen. Davon abweichend war die Speziesdifferenzierung von Keimen der Ordnung Enterobacteriales. Die beste Korrelation erreichte die Ordnung Clostridiales. Stämme ohne entsprechendes, dokumentiertes Korrelat in der Vitek MS-Datenbank (n=62) wurden ebenfalls in die Betrachtung mit eingeschlossen. Bei 31 Isolaten konnte kein Ergebnis ermittelt werden, 21 wurden massenspektrometrisch dem gleichen Genus zugeordnet, 10 Genusdifferenzierungen wichen ab. 315 Stämme wurden sowohl im Standardverfahren durch Konjugation von 1µl Ready-to-use Matrix als auch mit einem zweistufigen Säureextraktions- und Matrixkonjugationsverfahren gemessen und mit der solitären Konjugation der doppelten Matrixmenge verglichen. Die Abweichungen auf Genus- wie Speziesebene zwischen dem angewandten Standardprotokoll und den beiden anderen Verfahren waren deutlich signifikant. Im Vergleich der zweifachen Matrixmenge und dem zweistufigen Verfahren zeigte sich kein signifikanter Unterschied. Aufgrund der Ergebnisse scheint jedoch die Säureextraktion Gram-positiver Kokken in der Genusdifferenzierung von Vorteil zu sein, sie erreichte jedoch kein ausreichendes Signifikanzniveau. Die Daten sprechen eher dafür, eine Optimierung des Probe-Matrix-Verhältnisses anzustreben, zum Beispiel im Rahmen eines ausgedehnten Anwendertrainings. / Mass spectrimetric identification of bacteria by Vitek MS Routine lab System (IVD)
27

Chemical oxidation of tryptic digests to improve sequence coverage in peptide mass fingerprint protein identification

Lucas, Jessica Elaine 30 September 2004 (has links)
Peptide mass fingerprinting (PMF) of protein digests is a widely-accepted method for protein identification in MS-based proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is the technique of choice in PMF experiments. The success of protein identification in a PMF experiment is directly related to the amount of amino acid sequence coverage. In an effort to increase the amount of sequence information obtained in a MALDI PMF experiment, performic acid oxidation is performed on tryptic digests of known proteins. Performic acid was chosen as the chemical oxidant due to the ease of use and to the selective oxidation of cysteine, methionine, and tryptophan residues. In experiments performed in our laboratory, performic acid oxidation either increased or did not affect protein sequence coverage in PMF experiments when oxidized tryptic digests were analyzed by MALDI. Negative mode MALDI data were acquired, as well as positive mode MALDI data, due to the enhanced ionization of cysteic acid-containing peptides in negative mode. Furthermore, the confidence in a protein match is increased by observation of mass shifts indicative of cysteine, methionine, and/or tryptophan in oxidized peptide ion signals when comparing MALDI spectra prior to performic acid oxidation and after oxidation due to the low abundance of these residues in the majority of all known and hypothetical proteins.
28

Supported Lipid Bilayer Electrophoresis: A New Paradigm in Membrane Biophysics and Separations

Pace, Hudson 1982- 14 March 2013 (has links)
The motivation of this work was to produce novel analytical techniques capable of probing the physical properties of the cell surface. Many researchers have used supported lipid bilayers (SLBs) as models to study the structure and function of the cell membrane. The complexity of these models is consistently increasing in order to better understand the myriad of physiologically relevant processes regulated by this surface. In order to aid researchers in studying such phenomenon, the following contributions were made. To manipulate components within the cell membrane, an electrophoretic flow cell was designed which can be used as a probe to study the effect of electrical fields on charged membrane components and for the separation of these components. This devise allows for the strict control of pH and ionic strength as species are observed in real-time using fluorescence microscopy. Additionally, advancements have been made to the production of patterned heterogeneous SLBs for use in separations and to probe the interactions of membrane components. The methodology to couple SLB separations and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) imaging was devised. This technology allows for the label-free mapping of the SLB surface post electrophoresis in order to observe naturally occurring species unperturbed by the addition of extrinsic tags. The final contribution, and perhaps the greatest, is the development of a procedure to create highly mobile SLBs from native membranes. These surfaces have vast potential in that they are no longer simple models of the cell surface, they are in fact the actual cell surface made planar. This advancement will be of great use to biophysicists and biochemists interested in using surface specific analytical methods to better understand physiological processes. These highly mobile native membrane surfaces have been coupled with the SLB electrophoresis technology to separate discrete bands of lipids and proteins, a proof of principle that will hopefully be further developed into a standard method for membrane proteomic studies. Collectively the tools and methodologies described herein show great potential in allowing researchers to further add to mankind’s understanding of the cellular membrane.
29

An On-Target Performic Acid Oxidation Method Suitable for Disulfide Bond Elucidation Using Capillary Electrophoresis - Mass Spectrometry

Williams, Brad J. 2010 May 1900 (has links)
Disulfide bonds play important roles in establishing and stabilizing three-dimensional protein structure, and mass spectrometry (MS) has become the primary detection method to decipher their biological and pathological roles. Several experimental methods before or after MS detection have been developed to aid in disulfide bond assignment, such as tandem MS followed by database searching or modification of the disulfide bond via chemical reduction or oxidation. Despite these technological advancements, the detection and proper assignment of disulfide bonds have remained experimentally difficult. Therefore, we have developed an alternative method for disulfide bond elucidation using capillary electrophoresis-mass spectrometry (CE-MS) combined with an on-target performic acid oxidation method for matrix assisted laser desorption/ionization (MALDI) deposited samples. An information rich CE-MS method that results in distinct charge-state trends observed in two-dimensional plots of log(mu eff) versus log (MW) was developed to enhance the confidence of peptide and protein identifications. The charge-state trends provide information about the number of basic amino acid residues present within each peptide. This information can be used to develop methods to screen for posttranslationally modified peptides (e.g., phosphorylation, disulfide bonds, etc.). In the case of disulfide bonds, the highly charged peptides (i.e., 3, 4 or greater charge states) have a high probability of being disulfide-linked peptides, owing to charge contribution of both peptides forming the disulfide bridged peptide. However, intra-linked disulfide bridged peptides can also be present at lower charge states. Therefore, a chemically selective method to rapidly locate disulfide-linked peptides that have been separated by CE-MS must be developed. An on-target performic acid oxidation method was developed to provide the chemical selectivity towards disulfide bonds, i.e., converting the cystine bond to form two peptides modified with a cysteic acid (SO3H) side chain. The on-target oxidation method offers (i) no post-oxidation sample cleanup, (ii) improved throughput over solution-phase oxidation methods, and (iii) easily adapted to CE separations coupled offline with MALDI-MS. The evaluation of the on-target oxidation experimental parameters, the fragmentation behavior of cysteic acid-containing peptides and an alternative method for disulfide bond elucidation, using CE-MS combined with the ontarget oxidation method, are discussed within.
30

Synthese und Funktionalisierung linearer und zyklischer aromatisch-aliphatischer Aminoketone vom MICHLERs Keton-Typ

Anders, Susann 05 May 2010 (has links) (PDF)
In der vorliegenden Arbeit wird die Synthese linearer und zyklischer Aminoketone via nucleophiler aromatischer Substitution von fluorsubstituierten aromatischen Ketonen mit sekundären, aliphatischen Diaminen vorgestellt. Durch eine Adaption der Prozessparameter konnte eine elegante Methode zur Synthese fluorendgruppentragender Oligomere sowie von definierten Makrozyklen entwickelt werden. Die Modifizierung der Oligomere erfolgte sowohl durch Endgruppensubstitution als auch durch Reaktionen an der Carbonylgruppe am Oligomerrückgrat. Als Funktionalisierungsreagenzien wurden Mercaptoessigsäure, LAWESSONs Reagenz und N,N-Dimethylanilin eingesetzt. Die Umsetzung der Makrozyklen mit N,N-Dialkylanilinen ermöglicht die Synthese zyklischer Triphenylmethanfarbstoffe. Die Untersuchung der optischen Eigenschaften dieser zyklischen Kristallviolett-Derivate in Abhängigkeit des pH-Wertes und der Natur des Lösungsmittels sowie der Sensitivität gegenüber Cyanid-Ionen erfolgte mit Hilfe der UV/Vis-Spektroskopie.

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