Peanut skin, a low-value agriculture waste product, has drawn lots of research interest in recent years, due to its high content of A-type proanthocyanidins. A-type proanthocyanidins have been believed to contribute to cranberries' anti-UTI (urinary tract infection) effect. In this study, we compared the A-type proanthocyanidins in cranberry and peanut skin crude extracts using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Many similarities were found in the proanthocyanidin composition of cranberries and peanut skins. New oligomeric and polymeric proanthocyanidins in peanut skins, including heteroproanthocyanidins and proanthocyanidins with sugar moieties or galloyl esters, were tentatively identified. Solid phase extraction (SPE) and HPLC fractionation largely improved MALDI-TOF's ability to detect proanthocyanidins with high degrees of polymerization (DP). By analyzing the identified compounds in each fraction, we were also able to find some interesting elution pattern of the proanthocyanidins on the SPE cartridges and on the HPLC column. For example, the elution order on both the SPE cartridges and the diol phase column generally followed the DP. A-type proanthocyanidins tended to elute earlier than the B-type. Prodelphinidins retained much longer than other proanthocyanidins with the same DP. These findings may help researcher to identify future research directions and develop new separation methods to facilitate the identification of bioactive components in proanthocyanidin-rich plant extracts. / Ph. D.

The aim of this study was to optimize the sample preparation and methods for analysis of oligosaccharides of hyaluronic acid with MALDI- TOF-MS. The analysis was carried out on an Autoflex speed MADLI-TOF- MS instrument with both linear and reflectron mode. Matrices used in this study were 2,5-DHB and Super-DHB and type of matrix was chosen depending on the size of the analyzed oligosaccharides. The application of sample and matrix on the target that gave the most homogenous crystallization was sandwich and the laser power in the MALDI was kept at 65 %. Since it is known that salts and buffers interfere with the analysis, sample clean-up such as solid phase extraction (SPE) in pipette tips and dialysis was performed. SPE worked best for low mass oligosaccharides and provided high intensity and little noise. With SPE a concentration of the analyte could be done which was the advantage over dialysis. Dialysis worked well for larger oligosaccharides and mixtures of different sized oligosaccharides. Another way of using MALDI for biomolecule analysis is with TLC-MALDI. A fast and accurate separation was achieved and analysis could be done directly from the plate. The optimized methods were evaluated according to linearity and precision, LOD, mass accuracy and matrix stability. The linearity and precision was good in a higher concentration range (50 μg/mL and higher), but the test for limit-of-detection (LOD) indicated that concentrations from 20-30 μg/mL could be analyzed with no interference from the background. The mass accuracy was within the acceptable limits according to Bruker Daltonics when a mass calibration was done for each analyzed sample. The stability of the matrix in solution was difficult to study because of the day-to-day variation in intensities given by the MALDI-TOF-MS technique.

La synthèse peptidique non ribosomiale est réalisée par de grands complexes multienzymatiques, appelés synthétases, qui permettent la synthèse de peptide par une voie indépendante de l’intervention des ribosomes. Les peptides produits par cette voie de synthèse (NRPs) sont largement étudiés et divers outils d’analyse bioinformatique qui permettent la prédiction de la structure d’une synthétase ainsi que de la composition de son produit, sont disponibles. Ce type de synthèse peptidique a été décrit chez plusieurs microorganismes. Notre choix de modèle d’étude s’est porté sur les Pseudomonas fluorescents producteurs de deux types de NRPs, les lipopeptides cycliques (CLPs) et des sidérophores dont le plus représenté est la pyoverdine. L’étude de ces NRPs a été entreprise par des études expérimentales et bioinformatiques. Ces travaux ont permis de montrer le potentiel de synthèse non ribosomiale par une étude bioinformatique des 20 génomes de Pseudomonas disponibles. L’étude des synthétases de pyoverdine et l’extraction des signatures des domaines d’adénylation (A) ont permis d’améliorer la prédiction issue des outils d’analyse disponibles. Des expériences de feeding suivies par spectrométrie de masse MALDI-TOF ont permis de mettre en évidence des domaines A permissifs au sein des synthétases de pyoverdines. / The non-ribosomal peptide synthesis is performed by large multienzymatic complexes, called synthetases, which allow the synthesis of a peptide by a way independent of the intervention of the ribosomes. The peptides produced by this synthetic way (NRPS) are widely studied and various bioinformatics analysis tools that allow the prediction of the structure of the synthetase and the composition of his product, are available. This kind of peptide synthesis has been described in several microorganisms. Our choice focus on the fluorescent pseudomonads producing two types of NRPS, the cyclic lipopeptides (CLPS) and siderophores, the most represented is the pyoverdin. The study of these NRPS was performed by experimental and bioinformatics analysis. This work has demonstrated the potential of non-ribosomal synthesis by a bioinformatics study of the 20 avalaible genomes of Pseudomonas. Study of pyoverdine synthetases and extracting signatures of adenylation domains (A) have allowed to improving the prediction of the avalaible tools. Feeding experiments followed by MALDI-TOF helped to highlight permissive A domains in pyoverdins synthetases.

Fc-fusion proteins (e.g. EPO-Fc) are the most often created fusion proteins due to their beneficial biological and pharmacological properties. The economic success of Fc-fusion proteins and other biopharmaceuticals production however, greatly depends on a robust, low-cost and highly effective protein mammalian cell extraction system . Understanding of how cells respond to a protein production environment based on metabolic profiles provides new goals for bioengineering of cell lines for best performance in biomanufacturing. Furthermore, insights on how individual cell metabolism and therefore phenotype, respond to cell microenvironment allows the underlying biological mechanisms to be explored in greater detail. This study focused on the application of mass spectrometry (MS) technologies, combining the analysis of metabolic profiles of cells extracts by GC-MS and MALDI-MS and spatial visualisation and distribution of metabolites within cells producing the fusion protein by MALDI-MSI and SIMS imaging. The analysis of external and internal metabolome profiles of cells producing the protein showed an extended effect of EPO-Fc fusion protein production on cell metabolism. The findings indicate that changes observed in EPO-Fc producing cells are related to enhanced protein and lipid synthesis highlighting that these cells are in a state of increased metabolic activity with the protein exocytosis into growth medium. Moreover, the composition of lipid bilayer of induced cells seemed to be different to non-induced cells. These findings were confirmed with the analysis of EPO-Fc induced cells using MS metabolic imaging. Multivariate analysis highlighted a number of metabolites that were significantly influenced by the protein expression when compared to control cells. The major metabolic changes in induced cells were those related to lipid metabolism. The information about metabolic changes in tetracycline-induced cells obtained from the analysis of cell populations was further supported with the analysis based on single-cell studies. Single-cell based studies also proved that investigations of individual cells provide additional insights about changes in metabolism of induced cells that can be referred to a unique, single cell and its phenotype. The analysis of CHO cells revealed a high level of heterogeneity within a cell population. Different cell phenotype and hence, metabolite content allowed for correlation between cell locations and their metabolite characteristics, specific for each type of cells. This project has successfully shown combination of bio-analytical techniques to investigate external and internal metabolome changes related to a fusion protein production in mammalian cells. Additionally, the significance of single cell approaches in metabolomics has also been highlighted, providing insights into the sub-cellular distribution of metabolites in cells producing EPO-Fc and information on the level of heterogeneity within a cell population. A multidimensional approach for metabolic profiling and future technological improvements of single-cell platforms are required to provide improved data acquisition and data analysis in order to better understand unknown processes involved in cell metabolism.

Kohlenhydrat-Protein-Wechselwirkungen sind häufig entscheidend beteiligt an verschiedenen einer Infektion oder malignen Erkrankung zugrunde liegenden molekularen Erkennungs-prozessen, die zu Adhäsion, Zell-Zell-Interaktion sowie Immunreaktion und -toleranz führen. Trotz der hohen Relevanz für Diagnostik und Therapie dieser Erkrankungen sind die betreffenden Strukturen und Mechanismen bisher nur ungenügend untersucht und verstanden. Ziel dieser stark interdisziplinär angelegten Arbeit war es daher, Methoden der Fachbereiche Chemie und Pharmazie, Biologie und Medizin, aber auch Physik zu kombinieren, um Kohlenhydraterkennungsprozesse im Detail zu untersuchen und auf dieser Basis strukturell neuartige diagnostische und therapeutische Anwendungen zu entwerfen. Die hochkomplexe Zusammensetzung einer Zelloberfläche wurde zunächst auf ihren Glycan-anteil reduziert und stark vereinfacht auf der Oberfläche sogenannter Glycochips imitiert. Die verwendeten Systeme auf Basis einer Gold- bzw. Glasoberfläche ergänzen sich optimal in ihrer Eignung für komplementäre analytische Methoden wie Massenspektrometrie sowie quantifizierbare Fluoreszenzspektroskopie. Der Übergang auf die lebende Zelloberfläche gelang mit Hilfe des Metabolic Glyco-engineering, das die kovalente Präsentation definierter Motive durch eine Cycloaddition zwischen zwei bioorthogonalen Reaktionspartnern (z.B. Azid und Alkin) ermöglicht. Auf diese Weise wurden in Zusammenarbeit mit der Arbeitsgruppe Sauer (Universität Würzburg) zunächst die Dichte und Verteilung verschiedener Oberflächenglycane auf humanen Zellen mittels hochauflösender Fluoreszenzmikroskopie (dSTORM) bestimmt. Diese Parameter zeigten im Modell des Glycochips einen entscheidenden Einfluss auf Bindungsereignisse und multivalente Erkennung und zählen auch auf natürlichen Zelloberflächen – in engem Zusammenhang mit der lateralen und temporalen Dynamik der Motive – zu den wichtigen Faktoren molekularer Erkennungsprozesse. Die gezielte Modifikation zellulärer Oberflächenglycane eignet sich aber auch selbst als Methode zur Beeinflussung molekularer Wechselwirkungsprozesse. Dies wurde anhand des humanpathogenen Bakteriums S. aureus gezeigt, dessen Adhäsion auf Epithelzellen der Blasenwand durch Metabolic Glycoengineering partiell unterdrückt werden konnte. In einem ergänzenden Projekt wurden zwei potentielle Metabolite eines konventionellen Antibiotikums – des Nitroxolins – mit bakteriostatischer sowie antiadhäsiver Wirksamkeit dargestellt. Diese dienten als Referenzsubstanzen zur Verifizierung der postulierten Struktur der Derivate, werden aber auch selbst auf ihr Wirkprofil hin untersucht. Gleichzeitig stehen sie zusammen mit der Grundverbindung zudem als Referenz für die Wirkstärke potentieller neu entwickelter Antiadhäsiva zur Verfügung. / Interactions between carbohydrates and proteins often are crucial factors in the molecular recognition processes of infectious diseases or cancer, leading to adhesion and cell cell interaction, as well as immune response and immune tolerance. Despite of their high pertinence for diagnostics and successful therapeutic treatment of those diseases, the structures and mechanisms involved are still insufficiently studied and poorly understood. So it was the aim of this strongly interdisciplinary oriented dissertation, to study carbohydrate recognition processes on molecular basis and in detail by combining methods from different scientific schools like chemistry and pharmacy, biology and medicine, as well as physics. Based on the achieved results innovative diagnostic and therapeutic applications should be proposed. Initially the highly complex structural composition of a living cells’ surface was reduced to its’ glycan fraction and mimicked on the surface of so-called glycochips in a very simplified manner. Two systems, based on gold and glass surfaces respectively, were used due to their complementary applicability for different analytical methods like mass spectrometry and quantitative fluorescence spectroscopy. The step forward towards living cell surfaces was achieved by metabolic glycoengineering, a method that enables the covalent installation of defined binding motifs by performing a cycloaddition between two bioorthogonal reaction partners (e.g. azide and alkyne). In cooperation with the group of Prof. Dr. Markus Sauer (Universität Würzburg) this technique was used to determine the density and spatial distribution of different cell surface glycans on human cell lines with high resolution fluorescence microscopy (dSTORM). In the glycochip model these parameters exhibited a key value for binding processes and multivalent recognition. Even on native cell surfaces they are crucial factors of molecular recognition processes – closely related to the lateral and temporal dynamics of the structural motifs. In addition the specific modification of cell surface glycans itself can be used to manipulate molecular interaction processes. This could be shown for the human pathogenic bacterium Staphylococcus aureus by significant reduction of its adhesion potential towards epithelial cells derived from the human bladder after metabolic glycoengineering. Finally a supplementary project aimed to synthesize two prior postulated metabolites of the conventional antibiotic agent nitroxoline that shows bacteriostatic as well as antiadhesive effects. They were used as reference compounds to verify the postulated structure of the two derivatives and currently undergo further studies concerning their intrinsic mode of action. In addition to the parent molecule they also serve as reference compounds to estimate the potential of novel antiadhesives.

Sample preparation and analysis for atmospheric pressure matrix assisted laser desorption ionization time of flight mass spectrometry (AP- MALDI-TOF-MS) was investigated. By investigating the effects that sample preparation has upon MALDI signal, better analysis can be carried out. The influence of sample deposition was studied by not only observing the signal intensity produced but also by quantitation. Isotope dilution mass spectrometry (IDMS) was used for the quantitation of three different analytes. The results indicated that not only was signal greatly affected by sample deposition but the effect on quantitation error was also statistically significant among the three different sample deposition techniques that were evaluated. <br>Components of sample preparation solution were studied using polyethylene glycol (PEG) and polystryrene (PS) of different weights. This study altered the amounts of matrix, analyte and cationizing agent that were used to make up each sample. Not only did the sample signal intensity greatly vary which had statistical significance but a shifting of the polymer sample peaks was also observed. This confirms that sample preparation is of extreme importance for MALDI analysis. <br>Carpet fibers, glutathione and cell wall extracts from the bacteria Staphylococcus Epidermidis were also studied by AP- MADLI-TOF-MS. These analytes were carefully studied to provide an accurate characterization of each. The diversity of the analytes studied highlights the incredible capabilities that MADLI possesses being able to analyze a range of analytes. Though the samples were diverse each one was able to be completely and comprehensively analyzed using AP-MALDI-TOF-MS. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation

This report demonstrates a UV-embossed polymeric chip for protein separation and identification by Capillary Isoelectric Focusing (CIEF) and Matrix Assisted Laser Desportion/Ionization Mass Spectrometry (MALDI-MS). The polymeric chip has been fabricated by UV-embossing technique with high throughput; the issues in the fabrication have been addressed. In order to achieve high sensitivity of mass detection, five different types of UV curable polymer have been used as sample support to perform protein ionization in Mass Spectrometry (MS); the best results is compared to PMMA, which was the commonly used plastic chip for biomolecular separation. Experimental results show that signal from polyester is 12 times better than that of PMMA in terms of detection sensitivity. Finally, polyester chip is utilized to carry out CIEF to separate proteins, followed by MS identification. / Singapore-MIT Alliance (SMA)

Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

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