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Thesis (MScMedSc (Pathology. Medical Microbiology))--Stellenbosch University, 2008. / The genus Mycobacterium is a group of acid–fast, aerobic, slow- growing organisms which include more than 90 different species. A member of this genus, Mycobacterium tuberculosis, belonging to the Mycobacterium tuberculosis complex (MTB), is the causative agent of tuberculosis (TB). This disease is currently considered a global emergency, with more than 2 million deaths and over 8 million new cases annually. TB is the world’s second most common cause of death after HIV/AIDS. About one-third of the world’s population is estimated to be infected with TB. This catastrophic situation is further compounded by the emergence of Multi Drug Resistant tuberculosis (MDR-TB) and in more recent times, Extensive Drug Resistant tuberculosis (XDR-TB). Early diagnosis is critical to the successful management of patients as it allows informed use of chemotherapy. Also, early diagnosis is also of great importance if the menace of MDR-TB and XDR-TB is to be curbed and controlled. As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Traditional conventional laboratory procedures involving microscopy, culture and sensitivity tests may require turnaround times of 3-4 weeks or longer. Tremendous technological advancement over the years such as the advent of automated liquid culture systems like the BACTEC® 960 and the MGITTM Tube system, and the development of a myriad of molecular techniques most of which involves nucleic acid amplification (NAA) for the rapid identification of mycobacterial isolates from cultures or even directly from clinical specimens have contributed immensely to the early diagnosis of tuberculosis. Most of these NAA tests are nevertheless fraught with various limitations, thus the search for a rapid, sensitive and specific way of diagnosing tuberculosis is still an active area of research. The search has expanded to areas that would otherwise not have been considered ‘conventional’ in diagnostic mycobacteriology. One of such areas is mass spectrometry. This study joins the relatively few studies of its kind encountered in available literature to establish the ground work for the application of mass spectrometry, specifically Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) in the field of diagnostic mycobacteriology. This is an area which is in need of the speed, sensitivity and specificity that MALDI-ToF technique promises to offer. Since this technology is still in its infancy, the use of utmost care in the preparation of reagents, and the handling and storage of the organisms used to generate reference mass spectra for the database cannot be overemphasized. Similarly, the optimization of certain crucial experimental factors such as inactivating method and choice of matrix is of paramount importance. The main aim of this thesis was to generate a database of reference mass spectra fingerprints of selected (repository) Mycobacterium species. This necessitated the standardization of an experimental protocol which ensured that experimental factors and the various instrument parameters were optimized for maximum spectra generation and reproducibility. A standard operating procedure (SOP) for generating the database of reference mass spectra finger print of selected Mycobacterium species was developed and used to investigate the ability of the database to differentiate between species belonging to the same clinical disease complex as well as the nontuberculosis complex. The findings of this study imply that if the defined protocol is followed, the database generated has the potential to routinely identify and differentiate (under experimental conditions) more species of Mycobacterium than is currently practical using PCR and its related techniques. It is therefore a realistic expectation that when the database is clinically validated and tested in the next phase of the study, it will contribute immensely to the diagnosis of tuberculosis and other mycobacterioses. It will also aid in the identification of emerging pathogens particularly amongst the non-tuberculous mycobacteria.

Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to 'negatively' de-activate or 'positively' activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450's, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.

Plusieurs tests médicaux, comme celui du dépistage du cancer du sein, se basent sur l’observation de section tissulaire sous un microscope. Ces tests se basent sur l’interprétation d’un spécialiste et les résultats peuvent varier d’un expert à un autre dû la subjectivité des observations. L’utilisation d’une technique analytique offrant une quantification et une identification de cibles moléculaires dans une section tissulaire permettrait aux experts de produire des diagnostics plus objectifs et diminuerait possiblement le nombre de faux diagnostics. Les travaux présentés dans ce mémoire portent sur le développement d’une technique SPRi-MALDI-IMS permettant l’imagerie en deux dimensions de protéines contenues dans une section tissulaire. La MALDI-IMS est la technique de choix pour l’imagerie de biomolécules dans les sections tissulaires. Par contre, elle ne parvient pas à elle seule à quantifier de façon absolue le matériel adsorbé à la surface. Donc, le couplage de la MALDI-IMS avec la SPRi permet la quantification absolue de protéines en deux dimensions et crée une technique répondant aux besoins des experts médicaux. Pour ce faire, nous avons étudié, l’effet de la chimie de surface sur la nature et la quantité de matériel adsorbé à la surface du capteur. De plus, la cinétique de transfert des protéines du tissu vers le capteur a dû être optimisée afin de produire des empreintes correspondant au tissu d’origine, afin d’atteindre la gamme dynamique des instruments SPRi et MALDI-IMS. La technique résultante de ces optimisations permet d’obtenir les premières images quantitatives et qualitatives de protéines en deux dimensions d’une seule section tissulaire. / Several medical tests, such as breast cancer screening, are based on the observation of tissue section under a microscope. These tests rely on the interpretation of a specialist and the results can vary due to subjectivity of these analyses. Using an analytical technique able to quantify and identify molecular targets in a tissue section would enable experts to produce more objective diagnostic and possibly reduce the number of misdiagnosis. The work presented in this thesis focuses on the development of a SPRi-MALDI-IMS technique for imaging proteins in a tissue section. MALDI-IMS is the contemporary technique of choice for biomolecule imaging in tissue sections. However, absolute quantification of material absorbed to the surface cannot be performed using MALDI-IMS. The absolute quantification of proteins in two dimensions was successfully achieved by coupling of the MALDI-IMS with SPRi. Accordingly, we investigated by studying the nature of the surface chemistry and the amount of material adsorbed on the sensor’s surface. In addition, the kinetics of the protein transfer had to be optimized to produce imprints corresponding to the transferred tissue and to reach the dynamic ranges of both SPRi and MALDI-IMS instruments. The resulting technique led to quantitative and qualitative images of proteins in two dimensions of a single tissue section. It is envisioned that SPRi-MALDI-IMS can eventually meet the needs of medical experts for pathological screening.

This diploma thesis is focused on species identification of sand flies belonging to two genera of the subfamily Phlebotominae, genus Phlebotomus and Sergentomyia. Genus Phlebotomus together with the genus Lutzomyia of New World include the only proven vectors of Leishmania parasites and they are also carriers of viral and bacterial infections. Species of the genus Sergentomyia are proven vectors of sister genus Sauroleishmania that infects reptiles, but for several decades there have been speculations about their possible involvement in the transmission of mammalian Leishmania species. These suspicions arise mainly from repeated findings of mammalian Leishmania parasites in their digestive system. Correct species determination of medically significant hematophagous arthropods is very important especially for purposes of epidemiological studies so that efficient vector control may be correctly set. Routine identification of sand flies is based on morphological characters located mainly on their heads and genitalia. However, these characters may be variable within a species, they require certain expertise and in the field samples they may be damaged, making proper species identification impossible. This thesis therefore presents two alternatives of sand fly identification based on molecular...

En contrôlant le passage para- et transcellulaire des composés du sang vers le cerveau (et inversement), la barrière hémato-encéphalique (BHE) constitue la « gardienne » du compartiment cérébral. Bien que relativement connu dans son aspect physiologique, le phénotype BHE des cellules endothéliales des capillaires cérébraux (BCECs) reste mal compris au regard des mécanismes moléculaires qui gouvernent son établissement et son maintien. Dans cette optique, à l’aide du modèle in vitro de BHE développé au laboratoire (co-culture de BCECs bovines et de cellules gliales de rats), nous avons réalisé deux études protéomiques comparatives afin d’identifier les protéines cytoplasmiques potentiellement impliquées dans l’induction et le maintien de ce phénotype: d’une part une approche qualitative sans marquage (label free) et d’autre part une approche quantitative grâce à un marquage isotopique préalable des protéines (isotope-coded protein label, ICPL). Les deux approches, label free et ICPL se sont révélées complémentaires et ont permis, respectivement, l’identification de 447 et de 412 protéines (dont 290 quantifiées). Quatre protéines d’un intérêt particulier dans le domaine de la BHE (phosphatase alcaline tissu-non spécifique, TNAP ; protéine 1 possédant un domaine d’homologie à Eps15, EHD1 ; superoxyde dismutase, SODC et homologue 7 de la protéine de la maladie de Parkinson PARK7, DJ-1) ont fait l’objet de caractérisations biochimiques approfondies et ouvrent des pistes d’investigation sur des potentielles voies cellulaires induites par les cellules gliales et impliquées dans le phénotype BHE. / The blood-brain barrier (BBB) controls the para- and transcellular crossing of compounds from blood to brain (and inversely) and establishes the “gatekeepers” of the brain. The major part of therapeutic drugs developed to fight the brain diseases is deemed inefficient in vivo due to the presence of the BBB that they are unable to cross. Although relatively well known in its physiological aspect, the BBB phenotype of brain capillary endothelial cells (BCECs) remains largely under known and misunderstood in regards of the molecular mechanisms that govern its establishment and its maintenance. To this goal, using the in vitro BBB model developed in the laboratory (co-culture of bovine BCECs with rat glial cells), we performed two differential proteomic studies to identify the main cytoplasmic proteins involved in the establishment and maintenance of this phenotype: a qualitative label free approach and a quantitative isotope-coded protein labeling (ICPL) approach.The two different approaches, label free and ICPL, are complementary and led to the identification of 447 and 412 proteins, respectively. Four proteins of particular interest for BBB (tissue-non specific alkaline phosphatase, TNAP; Eps15 homology domain containing protein 1, EHD1; superoxide dismutase, SODC and Parkinson disease protein 7 homolog PARK7, DJ-1) have been more deeply studied and they open new discovery prospects related to cellular pathways induced by glial cells and involved in the BBB phenotype.

As mudanças climáticas associadas às atividades humana são devidas principalmente às emissões de CO2 na atmosfera provenientes da queima de combustíveis de origem fóssil. Desta forma, faz-se necessária a substituição dessas fontes fósseis de geração de energia, por fontes renováveis. Dentre as alternativas de fontes renováveis, podemos destacar os biocombustíveis produzidos a partir de microalgas, as quais apresentam composição rica em óleos e proteínas. Um dos grandes desafios encontrados na conversão de biomassa em biocombustíveis é a caracterização detalhada das microalgas. A identificação de espécies através da espectrometria de massas com Ionização/Dessorção à Laser Assistida por Matriz acoplada a analisador por tempo de vôo (MALDI-TOF-MS) utilizada na análise de perfil de proteínas de micro-organismos, e posterior rápida identificação por comparação com os padrões armazenados em bancos de dados (fingerprint) tem se sobressaído. Existem poucos trabalhos na literatura abordando a identificação de espécies de microalgas utilizando a técnica de MALDI-TOF-MS e nenhum trabalho abordando a análise a partir do uso de células de microalgas liofilizadas. Desta forma, nesse trabalho foi estudada a influência de diversos parâmetros tais como placa, modo de análise, valor de PIE, valor de IS2, matriz e solvente de matriz e amostra nos espectros de massas do tipo MALDI-TOF-MS para análise do perfil proteico de células liofilizadas das espécies de microalgas Chlorella vulgaris, Chlorella sp., Desmodesmus sp., Monoraphidium sp. e Oocystis sp. Primeiramente, os cultivos foram realizados em um sistema de agitador orbital otimizado de tal maneira que todas as posições apresentassem as mesmas condições. Após os cultivos, as células foram secas para posterior análise de espectrometria de massas. Para determinação da metodologia que fornecesse os melhores espectros de massas, foram avaliados, aleatoriamente, 3 parâmetros: número de íons (P1), relação sinal/ruído do pico base (P2) e intensidade do pico base (P3). Foi observado que para a maioria das amostras de microalgas, os parâmetros que mais influenciaram na obtenção de espectros de massas do tipo MALDI-TOF bem resolvidos foram a placa, o modo de análise, valor de PIE, valor de IS2 e a matriz. As variações obtidas nos espectros de massas, quando utilizados diferentes solventes tanto para a matriz quanto para a amostra, bem como a adição de isopropanol com o objetivo de melhorar a distribuição da matriz sDHB na placa de amostragem, não foram tão significativas como as observadas para os outros parâmetros avaliados nesse estudo. Como conclusão, o uso da matriz sDHB, solvente TA50 para amostra e matriz, análise na placa polished sob as condições de análise PIE 100ns, IS2 23kV mostraram-se muito mais efetivos para a análise de proteínas a partir de amostras de microalgas liofilizadas. A análise dos lipídios apresentou uma distribuição predominante dos ácidos graxos C16:0, C18:2 e C18:0 para os cultivos de 12 dias e C16:0, C18:2 e C22:6 para os cultivos de 8 dias. Entretanto, as proporções de C22:6 e C18:2 aumentaram para os cultivos de 8 dias. Dessa forma, as espécies de microalgas Chlorella vulgaris., Chlorella sp., Monoraphidium sp. e Oocystis sp. cultivadas por 8 dias podem ser convertidas em biocombustível por apresentarem ácidos graxos entre 14 e 18 carbonos e em sua composição. / Climate change associated to human activities are mainly due to CO&#8322 emissions from combustion of fossil fuels in the atmosphere. Thus, it is necessary to replace these fossil sources of energy generation for renewable sources. Among the alternative of renewable sources, biofuels derived from microalgae is am potential alternative, since microalgae present in their composition fatty acids and proteins. Characterization of microalgae is one of the challenges in the conversion of their biomass into biofuels. The microorganism species identification by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) thought the analysis of protein profile and subsequent fast identification by comparison with the standard protein profile (fingerprint) in the database has been outstanding. There are few studies in the literature about the identification of microalgae species using MALDI-TOF-MS technique and there is no one using cells of lyophilized microalgae. Thus, in this work was studied the influence of many parameters such as target, analysis mode, PIE, IS2 value, matrix, matrix solvent and sample solvent in the MALDI-TOF mass spectra for analysis of protein profile of lyophilized microalgae cells for the species Chlorella vulgaris cells, Chlorella sp., Desmodesmus sp., Monoraphidium sp. and Oocystis sp. Cultivations were carried out using an optimized shaker system, where all positions presented the same conditions. After the cultivation, the cells were dried for subsequent mass spectrometric analysis. To achieve the best mass spectra profile, 3 parameters were arbitrarily evaluated: number of ions (P1), base peak signal/noise ratio (P2) and base peak intensity (P3). It was observed for most microalgae samples, MALDI-TOF mass spectra profile were most influenced by target, analysis mode, PIE value, IS2 value and the matrix. Variations in the mass spectra obtained when different solvents were used (for matrix and sample) as well as the addition of isopropanol in order to improve the distribution of sDHB matrix on the spot, were not significant as that observed for the other parameters. In conclusion, the use of sDHB matrix, TA50 solvent for sample and matrix, analysis in polished target plate under the following analysis conditions: a PIE 100ns, a IS2 23kV, provided to be more effective for the analysis of protein from lyophilized microalgae cells. Lipid analysis of 12 days cultivated microalgae showed a predominant distribution of the C16:0, C18:2 and C18:0. The 8 days cultivation presented a distribution of C16:0, C18:2 and C22:6, but with C22:6 e C18:2 in a higher proportion. Since biofuel are produced by using the C14-C18 fatty acid contained in their composition, 8 days cultivation showed to be most effective for this purpose.

Dans un contexte d’étude des altérations biologiques survenant après un impact sur le système nerveux central (SNC), ma thèse porte sur l’étude des modifications protéomiques et lipidiques survenant après une lésion du SNC. Une étude spatio-temporelle a été menée sur un modèle TBI de rat afin d'identifier des marqueurs spécifiques de la lésion. En utilisant le MALDI-MSI, nous avons effectués une reconstruction 3D du cerveau lésé 3 jours post-lésion et nous avons représentés les molécules lipidiques spécifiques à la lésion. Après, cette analyse est réalisé avec d’autres délais après l’impact: 1, 3, 7 et 10 jours. En parallèle, une analyse microprotéomique est réalisée sur des coupes de tissus dans une approche visant à corréler les modifications lipidiques et protéiques. Nos résultats ont permis d'identifier une famille de lipides, les acylcarnitines, exprimés dans le cortex lésé avec une intensité maximale à 3 jours post-impact. Les données de protéomiques ont montrés une régulation positive de l’expression de protéines liées à la maladie de Parkinson. Dans l’ensemble, nos résultats décrivent un lien entre le TBI léger et la maladie de Parkinson dès 3 jours après l’impact, avec un rôle possible de l’acylcarnitine. Cette même famille de molécules est aussi présente dans les lésions médullaires. Dans une approche thérapeutique, les résultats précédents ont montrés que la protéine RhoA est un candidat majeur dans SCI. Après avoir utilisé un inhibiteur de RhoA, une étude protéomique a été réalisée pour évaluer l’impact sur ces lésions. Les résultats montrent que les traitements in-vivo et in-vitro avec l’inhibiteur stimule la croissance neuritique et la régénération axonale. / In the context of studying biological alterations occurring post impact to the central nervous system, my thesis was focused on studying the proteomic and lipid changes occurring post injury to the brain and spinal cord. A fundamental spatio-temporal study was conducted on an open-head rat TBI model to identify potential injury-specific markers. Using MALDI MSI, we performed 3D reconstruction of the injured brain at 3 days after injury and depicted lesion-specific m/z lipid molecules. After, MALDI MSI was applied on the acute/sub-acute time frame post impact: 1 day, 3 days, 7 days, and 10 days. In parallel, a microproteomic analysis was carried out on tissue segments directly consecutive to the imaged ones in an approach to correlate both lipid and protein changes. Our results yielded the identification of a family of lipids, acylcarnitines, which are expressed within the injured cortex with maximum intensity 3 days post impact. These lipid molecules also were found to be expressed in the substantia nigra and microproteomics data showed an upregulation in expression of Parkinson’s related proteins. Taken altogether, our results depict a role of link between mild-TBI and Parkinson’s disease as early as 3 days post impact, with a possible role of acylcarnitine. This same family of molecules was also present in SCI. In a therapeutic approach previous results showed RhoA protein as a major candidate post impact in SCI. After using RhoA inhibitor treatment, a proteomic study was carried out to investigate its impact on SCI. The results showed that both in-vivo and in-vitro treatment with RhoA inhibitor stimulated neurite outgrowth and helped in axonal regeneration.

Effective analytical techniques need to be developed to characterize the products of lignin degradation experiments to be able to generate renewable products from lignin. Mass spectrometry is an valuable analytical approach for lignin characterizaion, but it is hindered by lignin’s poor ionization efficiency, especially in the positive ion mode. In this work, we attempt to improve lignin’s ionization by utilizing electrospray and laser desorption mass spectrometry coupled with the addition of cations and chemical derivatives. We confronted the ionization problem from both a top-down and bottom-up analytical approach by analyzing synthesized monomers, dimers, and polymers along with natural lignin extracts from switchgrass. We also utilized tandem mass spectrometry to sequence lignin dimers and determine their bonding motifs from their fragmentation patterns. We believe that resolving the ionization issues with lignin will open the door for easier and more efficient lignin break-down techniques and ultimately more accessible renewable products from lignin.

Glykane sind weitverbreitete Biomoleküle, die meist in Form von Glykokonjugaten, wie beispielsweise als Glykoproteine oder Glykolipide, vorliegen. Durch die Interaktion von Glykanen mit Glykan-bindenden Proteinen wird eine Vielzahl an biochemischen Prozessen ausgelöst, sowohl physiologischer, als auch pathologischer Art. Die Aufklärung der beteiligten Glykanstrukturen ist daher nicht nur wichtig für das Verständnis dieser Prozesse, sondern kann auch Hinweise auf verschiedene Erkrankungen geben. Die Identifizierung von Glykanstrukturen kann über verschiedene Wege erfolgen. In der instrumentellen Analytik spielt dabei vor allem die ESI- und MALDI Massenspektrometrie eine wichtige Rolle, da diese sowohl für Detektion, als auch Fragmentierung großer Biomoleküle geeignet sind. Um die Analyse von Zuckern mittels chromatographischer und massenspektrometrischer Methoden zu erleichtern, werden häufig Derivatisierungsreagenzien eingesetzt. Diese verringern die Polarität der Zucker und erleichtern die Detektion durch das Einbringen von Chromo- oder Fluorophoren. Zur Derivatisierung am reduzierenden Terminus von Glykanen und Zuckern eignen sich vor allem Aminierungsreagenzien oder Hydrazide. Hydrazide haben gegenüber anderen Derivatisierungsreagenzien den Vorteil einer einfachen, salzfreien Umsetzung, aus der ein stabiles Derivat mit geschlossenem terminalen Zuckerring hervorgeht. Für die vorliegende Arbeit wurde die Derivatisierung mit den neuen Hydrazid Reagenzien INH und BINH, sowie dem bereits von Dr. P. Kapková bearbeiteten BACH untersucht. Als Vergleich dienten die underivatisierten Kohlenhydrate, wie auch das standardmäßig eingesetzte Aminierungsreagenz 2-AB. Dabei sollte das Ver-halten verschiedener Zucker und Glykane in Bezug auf chromatographische Trennung, Signalintensität und Fragmentierung analysiert werden. Zunächst wurde die Umsetzung von Mono-, Di- und Trisacchariden mit den neuen Derivatisierungsreagenzien INH und BINH optimiert. Dadurch konnte bei beiden Substanzen die komplette Umsetzung der Zucker in ihre Derivate gewährleistet werden. Auch die Derivatisierung mit Hilfe der Mikrowelle konnte bei INH erfolgreich durchgeführt werden. Auf diese Weise ließ sich die Reaktionszeit, im Vergleich zu den im Thermo-mixer® benötigten 90 Minuten, auf 20 Minuten verkürzen. Aufgrund der großen Men-gen an Zucker und Derivatisierungsreagenz, die für die Umsetzung in der Mikrowelle nötig sind, war der Versuch jedoch nur für INH geeignet. Im nächsten Schritt wurde das Trennverhalten der verschiedenen Mono-, Di- und Tri-saccharid-Derivate auf RP-C18- und HILIC-Phasen untersucht. Bei den Monosaccha-riden konnte durch keines der Derivate eine vollständige Trennung auf einer der Pha-sen erreicht werden. Das beste Ergebnis wurde durch INH auf der HILIC-Säule erzielt, doch auch dort konnten die Epimere Glucose, Mannose und Galactose nicht vollstän-dig separiert werden. Die Trennung der Disaccharide Maltose, Cellobiose und Lactose konnte auf der HILIC-Phase mit allen Derivaten außer BACH erfolgreich durchgeführt werden, auf der RP-C18 erwies sich dagegen nur 2-AB als geeignet. Bei den Trisac-chariden 3'SLN und 6'SLN konnten sowohl underivatisierte Zucker, als auch sämtliche Derivate mittels HILIC getrennt werden. Auch auf der C18-Phase war eine Trennung der BINH, BACH und 2-AB-Derivate möglich. Des Weiteren konnte durch die Derivati-sierungen die Signalintensität gegenüber den underivatisierten Zuckern deutlich gesteigert werden. Nach ihrer Trennung lassen sich massegleiche Di- und Trisaccharide anhand des Fragmentierungsmusters unterscheiden. Während bei den underivatisierten Disaccha-riden Maltose, Cellobiose und Lactose die charakteristischen Fragmente nur schwach sichtbar waren, konnte mit Hilfe der Hydrazide INH, BINH und BACH die Differenzie-rung deutlich erleichtert werden. Die 2-AB-Derivatisierung zeigte dagegen keine Ver-besserung der Fragmentierungseigenschaften. Bei der Unterscheidung der Trisaccharide 3’SLN und 6’SLN waren ebenfalls sowohl underivatisierte, als auch Hydrazid-derivatisierte Zucker im Vorteil gegenüber den 2-AB-Derivaten. Die Derivatisierung der N-Glykane von Ribonuclease B und Ovalbumin führte bei der Analyse mittels MALDI-TOF zu einer deutlichen Steigerung der Sensitivität. Beispiels-weise ließen sich bei den Glykanen des Ovalbumins durch die Derivatisierungen drei zusätzliche Strukturen im Vergleich zu den nativen Glykanen detektieren. Auch das Fragmentierungsverhalten der Glykane am MALDI-TOF/TOF konnte mit Hilfe der Derivatisierungen erheblich verbessert werden. Besonders die Umsetzung mit BINH führte zu einer Vielzahl charakteristischer Ringfragmente, wodurch die Aufklärung der verschiedenen Glykanstrukturen deutlich vereinfacht wurde. Auch im Vergleich zu 2 AB zeigten die Hydrazid-Derivate sowohl bessere Fragmentierungseigenschaften, als auch eine einfachere Handhabung für die Messung mittels MALDI-MS. Eine weitere Möglichkeit zur Identifikation von Glykanstrukturen liegt in der spezifischen Bindung durch Lektine. Diese Untersuchung gibt des Weiteren auch einen Hinweis auf funktionelle Eigenschaften der Glykane. Dafür wird die hohe Affinität von Biotin-haltigen Derivatisierungsreagenzien zu Avidin und Streptavidin genutzt. Nach der auf diese Weise erfolgten Immobilisierung der Glykane können diese mittels spezifischer Lektine nachgewiesen werden. Die Eignung des neuen Derivatisierungsreagen-zes BINH für diese Zwecke wurde anhand eines Glykan-Arrays getestet. Dadurch ließ sich bestätigen, dass BINH-derivatisierte Glykane und Zucker sowohl in der Lage sind an Streptavidin zu binden, als auch durch Lektine nachgewiesen werden können. Daher kann davon ausgegangen werden, dass BINH grundsätzlich für den Einsatz in bio-chemischen Methoden geeignet ist. Zusammenfassend lässt sich sagen, dass die Derivatisierung von Kohlenhydraten mit INH, BINH und BACH zu einer deutlichen Verbesserung der Trenn- und Fragmentierungseigenschaften führten. Dadurch konnten Identifizierung und Strukturanalyse sowohl von kleinen Zuckern, als auch von Glykanen erleichtert werden. Im Vergleich zu dem Standard-Derivatisierungsreagenz 2-AB zeigten die Hydrazide nicht nur im Bereich der Fragmentierungen, sondern auch durch die einfachere Derivatisierungsreaktion wesentliche Vorteile. / Glycans are widely spreaded biomolecules, which are commonly presented as gly-coconjugates, e.g. glycoproteins or glycolipids. The interaction of glycans with glycan-binding proteins triggers numerous physiological and pathological processes in bio-chemistry. Therefore the determination of the participating glycanstructures is of immens interest for the understanding of such processes. The structures of the involved glycans may even provide evidence on several diseases. Identification of glycan structures can be performed by means of various techniques. Favorable techniques in analytical chemistry are ESI- and MALDI- mass spectrometry, since they can be used for the detection, as well as for the fragmentation of large bio-molecules. To simplify the analysis of carbohydrates by means of chromatographic and mass spectrometric methods, different derivatization reagents are available. The chemical modification of the sugars leads to a decrease in polarity and to an enhanced detection by coupling to chromo- or fluorophores. Derivatization at the reducing end of saccharides and glycans can be performed through reductive amination, or coupling to hydrazide reagents. The advantage of hydrazides in comparison to other derivatization reagents lies in a simple, salt-free reaction and results in a stable derivative with closed terminal sugar ring, as the reduction step is not necessary. The present work concentrates on the derivatization of sugars and glycans using the newly developed hydrazides INH and BINH, in addition to BACH, which was already used by Dr. P. Kapková [99]. The derivatives of those reagents were compared to the underivatized carbohydrates, as well as to the commonly used derivatization reagent 2 AB, in order to observe the characteristics related to liquid chromatography, signal intensity and fragmentation behavior. In the first step, the derivatization reaction of mono, di- and trisaccharides with INH and BINH was optimized, in order to ensure a complete transformation of the carbohy-drates into their derivatized equivalents. The derivatization with INH was also per-formed via microwave. In this way, the reaction time was reduced from 90 minutes us-ing the thermomixer®, to 20 minutes. Since this method required a high amount of sample, it was only performed with INH. Next, the chromatographic behaviour of the different saccharide derivatives was ana-lyzed by reversed phase and HILIC high performance liquid chromatography. In case of the monosaccharides, none of the mixtures of derivatives could be separated com-pletely on either of these columns. For HILIC the best effect was achieved with INH, even though glucose and its epimers, mannose and galactose could not be separated completely. On reversed phase, derivatization with BACH and 2-AB showed the best results for the separation of the monomers, but here as well the epimers were not dis-solved. The separation of the INH, BINH and 2-AB derivatives of the disaccharides maltose, cellobiose and lactose was performed successfully on HILIC, with RP-C18 only 2-AB was proved to be suitable. For the trisaccharides 3’SLN and 6’SLN the na-tive forms, as well as all of the derivatives were separated using HILIC. Even by using reversed-phase, the separation of the BINH, BACH and 2-AB derivatives was possible. Furthermore the signal intensity of the derivatized carbohydrates was considerably en-hanced compared to the native saccharides. The chromatography of sugars on HILIC phase showed presence of isomers (very probably the anomers) of the analyzed sug-ars. If the appearance of these isomers is not desirable, separation on reversed-phase can be applied. After separation, di- and trisaccharides of the same mass can be distinguished, based on their fragmentation pattern. Whereas the native disaccharides maltose, cellobiose and lactose showed only slight differences in fragmentation, the derivatization with INH, BINH and BACH increased the number of characteristic fragments. In contrast, with 2-AB no improvement was achieved. The discrimination between 3’SLN and 6’SLN was also simplified by derivatization with the different hydrazides. In contrast to the 2-AB derivatives, the native trisaccharides showed a high content of characteristic fragments as well. For the glycans of ribonuclease B and ovalbumin, the sensitivity was clearly improved through derivatization. In the case of ovalbumin, three additional structures were detected, compared to the native glycans. Regarding the fragmentation by means of MALDI TOF/TOF, the fragmentation behaviour of the glycans was considerably better after derivatization. Especially the BINH derivatives generated a high amount of characteristic cross-ring fragmentations. This way, the elucidation of different isomeric glycan structures was enhanced. Again, 2-AB had worse characteristics in terms of fragmentation behaviour and application properties, compared to the hydrazide derivatives. Another option for the investigation of glycan structures is the specific recognition by lectins. This kind of study also provides information on the functional properties of gly-cans. Biotinylated derivatives possess a high affinity to avidin and streptavidin. This feature is used to immobilise glycans on (strept-)avidin coated surfaces, so they can be further analyzed by specific lectins. Due to this, the suitability of BINH for functional studies was tested. In initial studies BINH derivatized glycans and sugars were con-firmed to bind to streptavidin and were recognized by lectins as well. Hence, it can be assumed that BINH derivatives are suitable for this kind of biochemical analysis. In summary, the derivatization of carbohydrates using INH, BINH and BACH improved the behaviour of the analytes during liquid chromatography and mass spectrometric fragmentation. Hence, identification and structural analysis of small saccharides, as well as glycans were considerably simplified. Compared to common derivatization reagents like 2-AB, the hydrazides showed significant advantages, not only in terms of mass spectrometric fragmentation, but also because of their simple derivatization reaction.