Дисертації: "Matrice MALDI"

1

Manuelli, Pascal. "Etude des mécanismes de la désorption : ionisation laser assistée par matrice." Metz, 1995. http://docnum.univ-lorraine.fr/public/UPV-M/Theses/1995/Manuelli.Pascal.SMZ9520.pdf.

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Ce mémoire sur les mécanismes de la désorption/ionisation laser assistée par matrice (MALDI) est le fruit de travaux réalisés au laboratoire de spectrométrie de masse et de chimie laser (LSMCL) de l'université de Metz. Cette thématique est apparue au laboratoire suite au développement de l'analyse des biomolécules par spectrométrie de masse et à l'expérience accumulée depuis 15 ans sur la désorption laser. Nous nous sommes attachés à décomposer les étapes constituant une analyse MALDI afin de mieux comprendre les mécanismes mis en jeu lors de ce type d'ionisation. Nous avons plus précisément, à l'aide de molécules modèles (nucléotides, peptides,), étudie l'interaction entre le faisceau laser et les matrices couramment utilisées. Ceci nous a permis de mettre en évidence le rôle important des petites molécules neutres (notamment le dioxyde de carbone) qui sont éjectées lors de l'expansion du nuage gazeux induit par irradiation laser. Dans ce contexte, nous avons étudié des dérivés et des complexes de la cyclodextrine. Cette partie nous a permis d'expliquer les phénomènes d'interaction entre la molécule cible et les molécules de matrice ainsi que l'influence de la longueur d'onde du laser ionisant. A l'aide de la technique MALDI, nous avons pu mettre en évidence des différences structurales et caractériser des complexes d'inclusion intacts
This report on the mechanisms of matrix assisted laser desorption ionization (MALDI) was realized in mass spectrometry laboratory at Metz'university. This research field appeared in the laboratory after the development of biomolecules analyses by mass spectrometry and the experience stored for fifteen years on "laser desorption". We decomposed the different states of a MALDI experiment for a better understanding of this ionization process. By using some model compounds (as peptides, nucleotides,. . . ) we have studied more precisely the interaction between a laser beam and the different matrices. This allow us to show the great role of small neutral molecules (e. G. Carbone dioxide,. . . ) which are ejected in the plume after the laser pulse. Then, some derivative and complexes of cyclodextrins have been studied. This part is necessary to explain first the interaction between matrix and target molecules and secondly the dependance on the laser wavelength. Thank's to the MALDI technique we can highlight both both structural differences in intact inclusion complexes characterization

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2

Jaber, Ali. "Matrices MALDI bithiophéniques spécifiques aux alcaloïdes : étude des mécanismes fondamentaux et applications." Thesis, Angers, 2017. http://www.theses.fr/2017ANGE0042/document.

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Mon travail de thèse a consisté à poursuivre le développement et l’application des matrices bithiophéniques spécifique aux alcaloïdes. Après l’optimisation d’un protocole efficace d’analyse, adapté à l’objectif de l’étude, la mise au point d’une méthode de dosage des alcaloïdes par MALDI dans des extraits végétaux et sans prétraitement préalable a été étudiée. Cette méthode a pu être validée en étudiant des alcaloïdes existant dans différents extraits des plantes toxiques. Ensuite, la synthèse et l’évaluation de nouveaux composés bithiophéniques ont été présentés de manière à évaluer les facteurs favorisant l’interaction avec les alcaloïdes. Ultérieurement, cinq nouvelles matrices intéressantes furent l’objet d’une étude plus détaillée. Sur la base des résultats obtenus, le dérivé fluoré F T3 s’avère le plus efficace. ll présente une meilleure sélectivité que la matrice courante CHCA vis-à-vis des alcaloïdes et plus performant pour analyser les alcaloïdes dans différents mélanges complexes tels que des extraits bruts de plantes, des insectes, et des solutions bio-actives commerciales (médicament et répulsif). À la fin, ces sont regroupés les résultats de l’étude des paramètres thermodynamiques de la matrice MT3P, ce qui permettra de proposer des hypothèses expliquant la sélectivité de la matrice bithiophéniques fonctionnalisée pour les alcaloïdes
My thesis work consisted of pursuing the development and application of bithiophenic maldi matrices specific for alkaloids. After optimization of an efficient analysis protocol adapted to the objective of the study, a method for the determination of alkaloids in vegetable extracts by MALDI was developed. This method was validated by studying many alkaloids existing in extracts of different toxic plants. Subsequently, synthesis and evaluation of novel bithiophenic compounds were presented in order to evaluate the factors favoring the interaction with alkaloids. Then, five matrices among the molecules tested and having produced interesting results are chosen and were the subject of a more detailed study. On the basis of the results obtained, the fluorinated derivative PFPT3P proves to be the most effective molecules. It has a better selectivity with respect to alkaloids than the current matrix HCCA and performs better to analyze these metabolites in different complex mixtures such as crude extracts of plants,insects and commercial bioactive solutions (drug and repellent). At the end, the results of the study of the thermodynamic parameters of MT3P matrix are grouped. This will make possible to propose hypotheses explaining the selectivity of the functionalized bithiophene matrices for alkaloids

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3

Lavigne, Damien. "Conception de surfaces de matrice extracellulaire analysables en spectrométrie de masse SELDI-TOF." Paris 7, 2008. http://www.theses.fr/2008PA077247.

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Notre projet a pour objectif de concevoir des surfaces de matrice extracellulaire (MEC) sur des chips analysables en spectrométrie de masse SELDI-TOF (Surface Enhanced Laser Desorption lonization Time Of Flight). La MEC est synthétisée directement par les cellules en culture sur les chips. Le décollement des cellules par un traitement permet ensuite d'obtenir une surface de MEC. Parmi les chips en aluminium commerciales, seules les surfaces de silice permettent la culture des cellules musculaires lisses (hCML) et endothéliales (HUVEC) humaines et d'obtenir de la MEC. La MEC de ces deux types cellulaires est différente comme le révèle leur analyse au SELDI-TOF. Néanmoins, l'opacité des chips en aluminium gêne l'observation des cellules. Nous sommes les premiers à utiliser des chips transparentes en polyméthylpentèn< (PMP) au SELDI-TOF. Comme les cellules ne peuvent être cultivées directement sur ces chips, nous avons déterminé des conditions de fonctionnalisation au plasma permettant la culture des hCML. Des améliorations sont maintenant nécessaires dans le dispositif de culture pour conserver la MEC sur la surface du PMP fonctionnalisé pendant le traitement des cellules. Les spots de MEC sur les chips analysables directement au SELDI-TOF fournissent une nouvelle gamme de surfaces chromatographiques qui permettraient de découvrir de nouveaux biomarqueurs. D'autres applications peuvent être envisagées comme l'analyse de la différentiation cellulaire, ou l'étude de l'activité protéolytique de fluides biologiques sur la MEC
The aim of our project is the conception of extracellular matrix (ECM) surfaces on arrays suitable for direct analysis by SELDI-TOF mass spectrometry. The ECM is produced by cells in culture on the arrays, then, specific treatments remove the cells. On commercially available arrays made of aluminium, only silica spots allow the culture of human smooth muscle cells (hSMC) and endothelial cells (HUVEC) and the obtention of their ECM. According to their analysis with SELDI-TOF, the ECM of hSMC and HUVEC are different Nevertheless, opacity of aluminium arrays makes the cell observation difficult We are the first showing the us< of transparent arrays made of polymethylpentene (PMP) for SELDI-TOF analysis. As cells can not be cultured directly on PMP, we functionalized PMP arrays by gas plasma treatment to allow cell culture. Improvements are now required on the cell culture System to prevent the removal of the ECM from the arrays during the cell detachment treatment. The surfaces of ECM on arrays can be either analyzed directly by mass spectrometry or be used as a biomimetic retention surface in order to discover new biomarkers contained in biological fluids ECM-arrays could also be useful to study cell differentiation and protease activities on ECM surface

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4

Xu, Zeyuan. "PTMomics : Microfluidics for post-translational modifications studies : application to glycoproteomics." Electronic Thesis or Diss., Université Paris sciences et lettres, 2023. http://www.theses.fr/2023UPSLS029.

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Les glycoprotéines jouent un rôle fondamental dans la vie. Des études récentes montrent qu'elle est largement présente dans les activités biologiques, telles que la liaison des récepteurs, la signalisation cellulaire, la réponse immunitaire, le repliement des protéines et l'action des hormones. Parallèlement, la glycosylation des protéines a été associée au pronostic et à l'évolution de maladies comme les cancers. Ainsi, elle devient un biomarqueur important, et est également significative pour le développement thérapeutique. La glycosylation mérite donc d'être étudiée en détail. La glycoprotéine acquiert sa fraction d'hydrate de carbone par un processus appelé modification post-traductionnelle (PTM). Ce qui est différent de la plupart des PTM, c'est qu'il existe plusieurs formes de glycosylation. Les glycosylations N- et O-liées sont parmi les types les plus courants. D'autres comme la C-mannosylation et la phosphosérine sont rares. Si l'on considère simplement la glycosylation liée à l'azote, on peut la diviser en types complexes, hybrides et à haute teneur en mannose, qui résultent d'une synthèse non dirigée par un modèle. Sur la membrane du RE, un oligosaccharide sur Dol-PP est transféré à Asn dans la séquence Asn-X-Thr/Ser (X n'est pas la proline). Ensuite, les glycosidases et les glycosyltransférases agissent pour éliminer le glucose et ajouter une fraction de sucre dans le RE et le Golgi. Leurs activités dépendent des espèces, des types de cellules, des protéines, des sites et de l'état physiologique de la cellule, ce qui donne lieu à divers glycanes N-liés. Et l'hétérogénéité sera un obstacle majeur à l'étude des glycoprotéines. Les informations sur l'occupation de plusieurs sites peuvent être compliquées par le faible rendement d'ionisation et la fragmentation (macro-hétérogène). Sur chaque site, de multiples glycoformes existent, ce qui entraîne une diminution du signal (micro-hétérogène). De nombreux efforts ont été investis pour résoudre ce problème. La spectrométrie de masse (MS) est maintenant progressivement appliquée à l'étude de la glycosylation des protéines pour sa sensibilité, sa vitesse et son haut débit. Le couplage avec un système de chromatographie liquide à haute performance (HPLC) est une combinaison largement adoptée. Dans le même temps, des glycosidases, par exemple l'endo H et la PNGase F, sont découvertes et utilisées pour la libération des glycanes. La microfluidique est apparue pour le traitement des échantillons, et les approches hybrides sont de plus en plus populaires et devraient être la solution. En conclusion, pour une étude approfondie de sa fonction, l'occupation du site, la composition, la quantité et d'autres informations sont essentielles. Un protocole d'analyse amélioré doit être mis en œuvre pour acquérir spécifiquement ces informations à haute sensibilité, ce qui constitue le point d'ancrage de l'étude glycoprotéomique
Glycoprotein acts as a fundamental undertaker of life. Recent studies show that it is widely found in biological activities, such as in receptor binding, cell signaling, immune response, protein folding, and hormone action. Meanwhile, protein glycosylation has been associated to prognosis and progress of diseases like cancers. Thus, it becomes an important biomarker, and as well as is significant to therapeutics development. Thus, glycosylation worth to be studied in detail. Glycoprotein gains its carbon hydrate moiety through a process so called post translational modification (PTM). What is different from most of the PTMs is that there are various forms of glycosylation. N- and O-linked glycosylation are among the most common types. Others like C-mannosylation and phosphoserine are rare. Merely looking at the N-linked glycosylation, it can be further divided into complex, hybrid, and high mannose types, which is a result of the non-template-driven synthesis. On the ER membrane, an oligosaccharide on Dol-PP is transferred to Asn in the sequence Asn-X-Thr/Ser (X is not proline). Then, glycosidases and glycosyltransferases act in to remove glucose and adding sugar moiety in ER and Golgi. Their activities depend on the species, cell types, protein, sites, and physiological state of the cell resulting in various N-linked glycans. And heterogeneity will be a major hurdle to study glycoprotein. Multiple sites occupancies information can be complicated by the poor ionization yield and fragmentation (macro-heterogenous). On each site, multiple glycoforms existed leading to even diminished signal(micro-heterogenous). Many efforts have been invested in to solve it. Mass spectrometry (MS) is now progressively applied to protein glycosylation study for its sensitivity, speed, and high throughput. The coupling with a high-performance liquid chromatography system (HPLC) is a widely adopted combination. In the meantime, glycosidase, for example, endo H and PNGase F are discovered and used for glycan release. Microfluidics emerged for sample treatment, and hybrid approaches are becoming increasingly popular and expected to be the solution. To conclude, for an in-depth study of its function, site occupancy, composition, quantity, and other information are essential. An improved analysis protocol should implemented to specifically acquire this information at high sensitivity, which is the anchor for glycoproteomic study

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5

Guerandel, Cyril. "Etude de la qualité du piégeage des matières organiques par la matrice cimentaire vis-à-vis de la lixiviation." Thesis, Metz, 2009. http://www.theses.fr/2009METZ030S/document.

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Dans le cadre de la qualité environnementale des matériaux, il est essentiel d'apporter la preuve que les matériaux à base de ciments adjuvantés ne relarguent pas ou peu de matières organiques lors de leur contact avec l'eau constituant une solution lixiviante. Les additifs organiques, tels que les agents de mouture et les superplastifiants, constituent deux classes d'adjuvants organiques utilisés de manière systématique dans la fabrication ou la formulation des matériaux cimentaires, notamment quand ils sont en contact avec l'eau potable (conduites et châteaux d'eau). Pour évaluer le piégeage de ces composés organiques par une pâte de ciment CEM I, cinq montages de lixiviation dynamique CTG-LEACHCRETE ont été mis en place et adaptés pour l'étude de pâtes de ciment formulées avec des adjuvants organiques. La seconde partie de ce travail a pour objectif de mettre au point des techniques analytiques sensibles pour la détection de traces des constituants du superplastifiant et de l'agent de mouture directement dans les produits de la lixiviation de pâtes de ciment (les lixiviats) grâce aux techniques de spectrométrie de masse MALDI-TOF et Py-THM-MS. Enfin, l'application du protocole global de "lixiviation dynamique couplée à la spectrométrie de masse" nous permet d'apprécier la présence des composés organiques suite à des essais de lixiviation de pâtes de ciment formulées avec de l'agent de mouture et du superplastifiant. Cette démarche nous a permis d'obtenir de nombreux résultats donnant des informations sur les mécanismes de piégeage des différents additifs organiques par une pâte de ciment
Evidence that materials used by the industry are not damageable for the environment has become a major issue. In cement industry, organic admixtures such as grinding aids or superplasticizers ar widely used. In particular, they constitute cementitious materials in concrete contacting water like in water pipes and water tower. It is therefore essential to test whether these organic coumpounds are enventually dissolved into water by leaching. In this aim, five different dynamic leaching tests were developed and applied to a CEM I cement paste formulated with organic admixtures. In paralell, highly sensitive analytical methods based on MALDI-TOF and Py-THM-MS mass spectrometry techniques were designed in order to detect traces of leached superplasticizers or grinding aids. The dynamic leaching tests coupled to mass spectrometry allowed us to detect the presence of organic compounds in the leachate, and to better understand the mechanisms involved in the trapping of additives into a cement paste

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6

Alves, Sandra. "Une échelle acido-basique pour une meilleure compréhension du rôle de la matrice dans la production d'ions multichargés : une approche thermo-cínétique de l'ionisation en mode MALDI." Paris 6, 2002. http://www.theses.fr/2002PA066005.

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7

Mallah, Khalil. "In depth systemic biology analysis of central nervous system injuries." Thesis, Lille 1, 2018. http://www.theses.fr/2018LIL1S108/document.

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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

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8

Crank, Jeffrey Aaron. "Ionic liquids MALDI-MS matrices and gas chromatography stationary phases /." [Ames, Iowa : Iowa State University], 2009. 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:3379180.

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9

Hellwig, Nils [Verfasser]. "Das Laserablationsverhalten von ionischen Flüssigkeiten verschiedener MALDI-Matrices / Nils Hellwig." Kiel : Universitätsbibliothek Kiel, 2012. http://d-nb.info/1025465121/34.

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10

Allwood, Daniel Anthony. "Characterisation and ionisation modelling of matrices in MALDI mass spectrometry." Thesis, University of Hull, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301477.

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11

Kirmess, Kristopher Michael. "Investigation of Primary Ion Formation Mechanisms in UV-MALDI-MS Using Excited State Dynamics of Common MALDI Matrices." OpenSIUC, 2015. https://opensiuc.lib.siu.edu/dissertations/1110.

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The motivation of this dissertation is to provide insight towards primary ionization mechanisms within MALDI mass spectrometry. Albeit MALDI-MS is an extensively used analytical technique, the mechanism in which primary ions are created is still under scrutiny. Two current models of primary ionization exist which claim to elucidate the ion formation mechanisms within MALDI. In this work, excited state dynamics of MALDI matrices are shown to play an important role in the ionization mechanism. Upon inspection of the thermodynamic properties of commonly used MALDI matrices, no correlation was observed when plotted against their respective analyte ion yields. However, the excited state singlet lifetimes of these matrices seem to correlate well with their respective analyte ion yields. In the broadest sense, this correlation further supports the fact that photophysical properties of the matrix should be included in current UV-MALDI models. Investigation of a claim which stated singlet energy pooling reactions were absent in the MALDI matrix 2,4,6-trihydroxyacetophenone (THAP) resulted in the discovery of a new energy pooling mechanisms. Characteristic of aromatic ketones such as THAP, intersystem crossing is an efficient process in solution, which gives way to fluorescence in the solid state. Triplet pooling mechanisms from two neighboring THAP molecules are proposed and appear to be dependent on the preparation solvent used. These triplet pooling reactions are thought to play an important role in the primary ion formation mechanism within MALDI. To further investigate the theory of triplet species playing a vital role in MALDI ionization, the internal heavy-atom effect was employed to determine the effect of the triplet species. MALDI mass spectra and excited state decays of these heavy-atom substituted matrices were collected to demonstrate the relationship between triplet species and analyte ionization efficiency. Gas-phase thermodynamics and absorption at 337 nm were also examined to determine if these properties affected the analyte ion signal observed in the MALDI mass spectrum. Using the information collected from the previous study, an advanced MALDI matrix is synthesized. Addition of covalently bound iodine to the gold standard matrix, α-cyano-hydroxycinnamic acid, should drastically improve the performance of the non-substituted matrix due to the increase in triplet species present for pooling reactions. Sample preparation methods in MALDI are examined as are the effects of crystal morphology on the overall signal observed in the mass spectrum. Exciton hopping and pooling rates are highly dependent on intermolecular interactions, so it is expected that crystal packing will affect MALDI. As noted for THAP, preparation solvent plays a significant role in not only crystal morphology, but also the excited state dynamics for all matrices studied.

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12

Jacksén, Johan. "Improved techniques for CE-MALDI-MS off-line coupling and MALDI-MS analysis of primarily hydrophobic proteins and peptides." Licentiate thesis, KTH, Chemistry, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4599.

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Due to the hydrophobic nature of integral membrane proteins (IMP) they give rise to several difficulties concerning handling and analysis, which is not the case for the most water soluble proteins. New analysis methods are needed, where the insolubility problems of the hydrophobic proteins due to aggregation and adhesion are tackled. Those problems also affect digestion performance and equipment compatibility for the analysis.

Protocols for analysis and separation specified for IMP are presented in Paper I and III.

The instrumentation used in this work was capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Both instruments are suitable for peptide/proteins analysis.

In Paper I, protocols for a CE separation of bacteriorhodopsin (BR) peptides as model IMP peptides are established. Also, a partially automated manufacturing procedure of a concentration MALDI-target is presented, suitable for fractions from CE. The MS analysis detected 9 out of 10 cyanogen bromide (CNBr) digested BR peptides. A novel technique for the off-line integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a microcanal functioning as a MALDI target window. Investigation of the microcanal electro-osmotic flow (EOF) properties and band broadening characteristics was performed. A protein separation was obtained and detected with MALDI-MS analysis in the microcanal. Different protein digestion methods were evaluated using BR in Paper III through MALDI-MS. Several digestion methods as well as MS media were investigated alongside different MALDI matrices. For example, matrices as the hydrophobic 2,6-dihydroxyacetophenone (DHAP) and 2-Hydroxy-3-methoxybenzoic acid (2H3MBA) or 2-Hydroxy-5-methoxybenzoic acid (2H5MBA) mixed with DHB, appeared to be promising matrices for analysis of BR.

Med anledning av integrala membranproteiners (IMP) hydrofoba egenskaper uppstår flera svårigheter vid hantering och analys av IMP, vilket inte är fallet för vattenlösliga proteiner. Nya analysmetoder krävs, som löser löslighetsproblemen för de hydrofoba proteinerna som tex flockning och adsorbtion. Dessa problem påverkar även klyvningsgrad och kompatibilitet med analysutrustningen.

I Artikel I och Artikel III presenteras protokoll för analys och separation specifikt för IMP. Instrumenteringen som har använts i detta arbete är kapillärelektrofores (CE) och matris-assisterad laserdesorptions-joniserings-masspektrometri (MALDI-MS). Båda instrumenten är lämpade för peptid/protein analyser.

I Artikel I, presenteras protokoll för en CE separation av peptider från bacteriorhodopsin (BR), som användes som modellpeptider för IMP. En delvis automatiserat tillverkningsprocedur för en koncentrerande MALDI-platta, som är anpassad för CE fraktionerna beskrivs också. MS-analysen detekterade 9 av 10 BR-peptider från cyanobromid-klyvning (CNBr). En ny teknik för off line-integrering av CE till MALDI-MS genom ett slutet-öppet-slutet system presenteras i Artikel II, där den öppna delen är en mikrokanal som fungerar som detektionsfönster i MALDI. Undersökning av mikrokanalens egenskaper som tex det elektroosmotiska flödet (EOF) och bandbreddningen utvärderades. En proteinseparation genomfördes och detekterades med MALDI–MS i mikrokanalen. Olika proteinklyvningsmetoder för BR undersöktes i Artikel III med MALDI-MS. Flera proteinklyvningsmetoder samt MS-medier utvärderades tillsammans med olika MALDI-matriser. Den hydrofoba matrisen 2,6-dihydroxyacetophenone (DHAP) och 2-Hydroxy-3-methoxybenzoic acid (2H3MBA) eller 2-Hydroxy-5-methoxybenzoic acid (2H5MBA) blandade med DHB, visade sig exempelvis vara lovande matriser för BR-analyser.

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13

Krüger, Ralf. "Untersuchungen zum Einbau von Analytionen in MALDI-Matrizes sowie zur Ionisation und Adduktbildung in der MALDI-Massenspektrometrie." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969681682.

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14

Bronzel, Júnior João Luiz [UNESP]. "Matrizes iônicas: detecção e quantificação de cianotoxinas por maldi-ms." Universidade Estadual Paulista (UNESP), 2015. http://hdl.handle.net/11449/143001.

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A técnica de ionização MALDI (Matrix-Assisted Laser Desorption/Ionization) foi uma das responsáveis por revolucionar a Espectrometria de Massas, tornando possível sua aplicação a moléculas termolábeis e de alta massa molecular, como as proteínas. Apesar de seu grande desenvolvimento, esta técnica ainda apresenta algumas limitações como a seleção da matriz utilizada e o método de preparo de amostras. Estes fatores são determinantes na reprodutibilidade da análise e na faixa de aplicação de m/z. Neste trabalho, inicialmente analisou-se diferentes linhagens de cianobactérias por MALDI-TOF-MS, com a finalidade de se buscar por novos analitos para os quais fosse possível o monitoramento utilizando-se as matrizes iônicas. Posteriormente avaliou-se as características destas matrizes, a metodologia de preparo de amostras, a composição do analito e a intensidade do laser da fonte de MALDI, com o objetivo de selecionar as melhores metodologias de análise. Após esta etapa, as matrizes iônicas e metodologias selecionadas foram aplicadas na detecção e quantificação de cianotoxinas e na análise de fármacos por MALDI-MS. Neste estudo obteve-se como resultados: a diferenciação de linhagens de cianobactérias através dos fingerprints obtidos por MALDI-TOF-MS que também permitiram a detecção de importantes metabólitos; a detecção da homoanatoxina-a, uma cianotoxina de baixa massa molecular, diretamente de células de cianobactérias; o desenvolvimento de um método quantitativo de análise de microcistinas com ótima precisão; e o desenvolvimento de uma metodologia inédita para a detecção dos componentes ativos de medicamentos, ampliando, portanto, o leque de aplicações da técnica de MALDI.
The Matrix-Assisted Laser Desorption/Ionization technique was one of the ionization sources responsible for revolutionizing Mass Spectrometry, making possible its application to labile and high molecular weight molecules, such as proteins. Despite its great development, this technique still has some limitations like the selection of the matrix and the sample preparation method. These factors determine the reproducibility of analysis and the m/z application range. Initially, in this work, we analyzed different cyanobacteria strains by MALDI-TOF-MS, in order to check for new analytes to be monitored using ionic matrices. Subsequently, the characteristics of these matrices, the sample preparation method, the composition of the analyte and laser intensity of the MALDI source were evaluated aiming to select the best methodologies of analysis. After this step, the selected ionic matrices and methodologies have been applied in the detection and quantification of cianotoxins and analysis of pharmaceutical drugs by MALDI-MS. In this study we obtained as results: the differentiation of strains of cyanobacteria through the fingerprints obtained by MALDI-TOF-MS which allowed the detection of important metabolites; the detection of homoanatoxin-a, a low molecular weight cyanotoxin, directly from cyanobacteria cells; the development of a quantitative method for the analysis of microcystins with great precision; and the development of a new methodology for the detection of active compounds of medicines, increasing, therefore, the application range of MALDI technique.

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15

Bronzel, Júnior João Luiz. "Matrizes iônicas : detecção e quantificação de cianotoxinas por maldi-ms /." Araraquara, 2015. http://hdl.handle.net/11449/143001.

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Orientador: Humberto Márcio Santos Milagre
Banca: Eduardo Maffud Cilli
Banca: José Augusto Rosário Rodrigues
Resumo: A técnica de ionização MALDI (Matrix-Assisted Laser Desorption/Ionization) foi uma das responsáveis por revolucionar a Espectrometria de Massas, tornando possível sua aplicação a moléculas termolábeis e de alta massa molecular, como as proteínas. Apesar de seu grande desenvolvimento, esta técnica ainda apresenta algumas limitações como a seleção da matriz utilizada e o método de preparo de amostras. Estes fatores são determinantes na reprodutibilidade da análise e na faixa de aplicação de m/z. Neste trabalho, inicialmente analisou-se diferentes linhagens de cianobactérias por MALDI-TOF-MS, com a finalidade de se buscar por novos analitos para os quais fosse possível o monitoramento utilizando-se as matrizes iônicas. Posteriormente avaliou-se as características destas matrizes, a metodologia de preparo de amostras, a composição do analito e a intensidade do laser da fonte de MALDI, com o objetivo de selecionar as melhores metodologias de análise. Após esta etapa, as matrizes iônicas e metodologias selecionadas foram aplicadas na detecção e quantificação de cianotoxinas e na análise de fármacos por MALDI-MS. Neste estudo obteve-se como resultados: a diferenciação de linhagens de cianobactérias através dos fingerprints obtidos por MALDI-TOF-MS que também permitiram a detecção de importantes metabólitos; a detecção da homoanatoxina-a, uma cianotoxina de baixa massa molecular, diretamente de células de cianobactérias; o desenvolvimento de um método quantitativo de análise de microcistinas com ótima precisão; e o desenvolvimento de uma metodologia inédita para a detecção dos componentes ativos de medicamentos, ampliando, portanto, o leque de aplicações da técnica de MALDI.
Abstract: The Matrix-Assisted Laser Desorption/Ionization technique was one of the ionization sources responsible for revolutionizing Mass Spectrometry, making possible its application to labile and high molecular weight molecules, such as proteins. Despite its great development, this technique still has some limitations like the selection of the matrix and the sample preparation method. These factors determine the reproducibility of analysis and the m/z application range. Initially, in this work, we analyzed different cyanobacteria strains by MALDI-TOF-MS, in order to check for new analytes to be monitored using ionic matrices. Subsequently, the characteristics of these matrices, the sample preparation method, the composition of the analyte and laser intensity of the MALDI source were evaluated aiming to select the best methodologies of analysis. After this step, the selected ionic matrices and methodologies have been applied in the detection and quantification of cianotoxins and analysis of pharmaceutical drugs by MALDI-MS. In this study we obtained as results: the differentiation of strains of cyanobacteria through the fingerprints obtained by MALDI-TOF-MS which allowed the detection of important metabolites; the detection of homoanatoxin-a, a low molecular weight cyanotoxin, directly from cyanobacteria cells; the development of a quantitative method for the analysis of microcystins with great precision; and the development of a new methodology for the detection of active compounds of medicines, increasing, therefore, the application range of MALDI technique.
Mestre

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16

Bouschen, Werner. "Ortsaufgelöste MALDI-Massenspektrometrie an biologischen und synthetischen Oberflächen." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971766886.

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17

Börjesson, Erik. "Undersökning av jonvätskematrisers detektion på olika typer av MALDI plattor." Thesis, KTH, Skolan för kemivetenskap (CHE), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-195847.

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Examensarbetet gjordes för att undersöka om det istället för 2,5-dihydroxidbensoesyra fanns en jonvätskematris som hade en lika bra eller bättre detektionsgrad. Examensarbetet undersökte också om det var någon detektions skillnad på stål- och koncentrationsplatta när analysmetoden matris-assisterande laser desorption-jonisation-masspektrometri (MALDI-MS) analysmetod används. Jonvätskematris är en matris som binder till en svag bas och är till för att skydda peptiderna från addukter. Baserna som undersöktes var etylamin, dietylamin, diisopropylamin och N,N-etyl-diisopropylamin. Examensarbetet jämförde matrisen med jonvätskematriserna som alla var upplösta i etanol, vatten eller en vätskeblandning (TA 30) (0,1 % trifluorättiksyra i vatten och acetonitril i 7:3 volym). Det utfördes tester genom att matrisen och jonvätskematriserna blandades med en peptidblandning innehållande åtta stycken peptider. Blandningen deponerades på en stål- och en koncentrationsplatta. Efter att blandningen hade torkat på plattorna och bilde till kristaller eller en vätskefilm placerades plattorna i MALDI-apparaturen. Testerna utvisade att N,N-etyl diisopropylamin blandningen i etanol och vätskeblandningen var bättre än 2,5-dihydroxybenzene på att detektera antalet peptider på koncentrationsplattan. Det visades att etylamin och dietylamin hade samma detektiongrad som 2,5-dihydroxidbensoesyra i etanol och vatten vid detektion av antal peptider på koncentrationsplattan. En detektionsskillnad fanns mellan stål- och koncentrationsplatta. Stålplattan var bättre på att detektera tyr-bradykinin. Vid vidare studier på området skulle testerna utföras på stålplatta för att detektera tyr-bradykinin och resterande peptider på koncentrationsplatta. Vid vidare studier skulle TA 30 och etanol som lösningsmedel användas och matriserna ska upplösas i mol-koncentration istället för vikt-koncentration, för att få en mer jämförbar detektion.
This degree project was made to examine if there was a better ionic liquid matrix instead of the matrix 2,5-dihydroxybenzene. The aim of the project was also to investigate if there were any detection differences between anchorchip and groundsteel plate when testing matrix assisted laser desorption ionization masspectrometry (MALDI-MS). An ionic liquid matrix is a matrix that binds to a base used to protect the peptides from adducts. In the degree project 2,5-dihydroxybenzene was compared with the ionic liquid matrices which is 2,5-dihydroxybenzene combined with a base. The bases were ethylamine, diethylamine, diisopropylamine and N,N- ethyldiisopropylamine. All matrices were dissolved in ethanol, water or a liquid mix (0,1 % Trifluoroacetic acid in water and acetonitril in 7:3 volume). The tests were performed by mixing the matrix and the ionic liquid matrices with a peptide mix which contained 8 types of peptides. The matrix/ionic liquid matrices and peptide mix was deposited on a ground steel and anchorchip MALDI plate. After the mixture had evaporated and transformed to crystals/film, the plates were put in MADLI equipment. The results showed that N,N- ethyldiisopropylamine in ethanol and liquid mix had better detection than 2,5-dihydroxybenzene. Ethylamine and diethylamine had the same detection as 2,5-dihydroxybenzene in ethanol on anchorchip plate. There were detection differences between the anchorchip and ground steel plate. In favor of ground steel plate when detecting tyr-bradykinin, but for detecting the rest of the peptides anchorchip is favorable. If further studies would be performed in this area it would be done in the liquid mix and ethanol as solvent. For more comparable detection results the matrices concentration should be in mol/L instead of g/L.

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18

Siricord, Cornelia Charito. "Detection of Phytophthora species by MALDI-TOF mass spectrometry." Thesis, Siricord, Cornelia Charito (2005) Detection of Phytophthora species by MALDI-TOF mass spectrometry. PhD thesis, Murdoch University, 2005. https://researchrepository.murdoch.edu.au/id/eprint/314/.

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Phytophthora diseases have caused worldwide economic, social and environmental impacts for decades. Once their presence is confirmed, they are difficult to eradicate. To reduce and manage the damage inflicted by the pathogen, fast and reliable disease management protocols are required. Tests that enable the rapid and reliable identification of the pathogen assist greatly in disease management. Phytophthora species are traditionally not only detected by baiting but also by plating of symptomatic tissue on selective media. Species can be identified by the characteristics of the mycelium growing out of the bait. However, the method is low throughput, labour intensive, and prone to false negatives. An alternative approach would be to detect the pathogen by the presence of its DNA. This involves amplification of the pathogen DNA using Polymerase Chain Reaction (PCR) and detection of the amplification product. Detection is usually by agarose gel electrophoresis. However, this is also a labour intensive process involving pouring, loading, running, and staining of the gels. The aim of this thesis is to explore the use of Matrix Assisted Laser Desorption/ Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry for detection of PCR products. This procedure enables the analysis of large numbers of samples within a very short time-frame as the average time for analysis of each sample is in the order of milliseconds. The assay involves annealing an extension (genotyping) primer to the PCR product and its extension by a single nucleotide. The nature of the nucleotide added differentiates species as does the site to which the primer anneals. Multiple extension (genotyping) primers can be used together in a single reaction for detection of multiple species. In this project four genotyping primers (GPs) were designed from the ITS regions of Phytophthora palmivora, Phytophthora cinnamomi, Phytophthora citricola, and Phytophthora cambivora. The extension primers were tested for their specificity on the DNA of the target species. The four primers designed were specific for their intended targets except for GPpalm3 which in addition to being extended by ddT when tested with DNA from P. palmivora, was also extended by ddC when tested with DNA from other species of Phytophthora or Pythium. These primers were also tested for their ability to detect multiple Phytophthora species in a single reaction (multiplexing). Mixtures of primers were added to mixed DNA templates and the primer extension reaction carried out. The primers were designed so that their masses were sufficiently different for them to be identified from a mixture. Six replicates were analysed for each reaction. In general only about 1-3 of the six replicates gave a positive reaction. This indicates that there may be some interference between primers, or that the presence of all four nucleotides interfered with the primer extension reaction. Increasing either the amount of enzyme, the amount of nucleotides or both did not improve the results. The sensitivity of detection was tested by the addition of different amounts of mycelium to soil. The detection sensitivity depended on the primer pair used for PCR amplification. The ITS1/2 primer pair was more sensitive than the ITS1/4 pair. The limit of detection was 1 mcg mycelium g soil-1. However using nested PCR, levels of sensitivity comparable to those obtained using the ITS1/2 primer pair could be achieved. Primers to other regions of the genome such as the beta cinnamomin elicitin gene gave very low levels of sensitivity compared to the ITS primers. In comparison with DNA detection we found that the limit of detection using baiting was 4 mcg mycelium g soil-1. Results below this limit were unreliable. The method suffered from the additional disadvantage that it took a long time in comparison to DNA detection. DNA detection methods do not distinguish between living and dead organisms in the soil. However it can be hypothesised that DNA is unlikely to persist for any significant length of time in soil. To test this, we added plasmid DNA to soil and tested the persistence of this DNA using a variety of methods such as precipitation of labelled DNA, southern blotting and PCR amplification. It was found that in general, in soils from different ecosystems, the bulk of the DNA was undetectable after 24 hours. The rate of DNA breakdown differed with the soil type. In some soils, the added DNA was not detected even after 2 hours, whereas in others it could be observed after 10 hours. The detection depended on the method. Southern blotting showed that although DNA could be observed at 10 hours, by 24 hours it was completely degraded. In contrast a PCR product could be obtained from the soil extracts up to 24 hours. In a separate experiment, plasmid DNA was detectable over a 24 hour incubation period in 5 soil samples from 5 different sites. The results suggest that DNA is degraded rapidly in soil and is unlikely to persist longer than 24 hours. The results in this thesis demonstrate that MALDI-TOF MS is a suitable alternative to agarose gel electrophoresis for analysis of PCR products. The technique is rapid, differentiates species from mixtures, is high-throughput and amenable to automation. Implementation will require further research to automate the primer extension assay to reduce the sensitivity to impurities in the DNA and to design parameters for sampling asymptomatic material.

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19

Siricord, Cornelia Charito. "Detection of Phytophthora species by MALDI-TOF mass spectrometry." Siricord, Cornelia Charito (2005) Detection of Phytophthora species by MALDI-TOF mass spectrometry. PhD thesis, Murdoch University, 2005. http://researchrepository.murdoch.edu.au/314/.

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Анотація:

Phytophthora diseases have caused worldwide economic, social and environmental impacts for decades. Once their presence is confirmed, they are difficult to eradicate. To reduce and manage the damage inflicted by the pathogen, fast and reliable disease management protocols are required. Tests that enable the rapid and reliable identification of the pathogen assist greatly in disease management. Phytophthora species are traditionally not only detected by baiting but also by plating of symptomatic tissue on selective media. Species can be identified by the characteristics of the mycelium growing out of the bait. However, the method is low throughput, labour intensive, and prone to false negatives. An alternative approach would be to detect the pathogen by the presence of its DNA. This involves amplification of the pathogen DNA using Polymerase Chain Reaction (PCR) and detection of the amplification product. Detection is usually by agarose gel electrophoresis. However, this is also a labour intensive process involving pouring, loading, running, and staining of the gels. The aim of this thesis is to explore the use of Matrix Assisted Laser Desorption/ Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry for detection of PCR products. This procedure enables the analysis of large numbers of samples within a very short time-frame as the average time for analysis of each sample is in the order of milliseconds. The assay involves annealing an extension (genotyping) primer to the PCR product and its extension by a single nucleotide. The nature of the nucleotide added differentiates species as does the site to which the primer anneals. Multiple extension (genotyping) primers can be used together in a single reaction for detection of multiple species. In this project four genotyping primers (GPs) were designed from the ITS regions of Phytophthora palmivora, Phytophthora cinnamomi, Phytophthora citricola, and Phytophthora cambivora. The extension primers were tested for their specificity on the DNA of the target species. The four primers designed were specific for their intended targets except for GPpalm3 which in addition to being extended by ddT when tested with DNA from P. palmivora, was also extended by ddC when tested with DNA from other species of Phytophthora or Pythium. These primers were also tested for their ability to detect multiple Phytophthora species in a single reaction (multiplexing). Mixtures of primers were added to mixed DNA templates and the primer extension reaction carried out. The primers were designed so that their masses were sufficiently different for them to be identified from a mixture. Six replicates were analysed for each reaction. In general only about 1-3 of the six replicates gave a positive reaction. This indicates that there may be some interference between primers, or that the presence of all four nucleotides interfered with the primer extension reaction. Increasing either the amount of enzyme, the amount of nucleotides or both did not improve the results. The sensitivity of detection was tested by the addition of different amounts of mycelium to soil. The detection sensitivity depended on the primer pair used for PCR amplification. The ITS1/2 primer pair was more sensitive than the ITS1/4 pair. The limit of detection was 1 mcg mycelium g soil-1. However using nested PCR, levels of sensitivity comparable to those obtained using the ITS1/2 primer pair could be achieved. Primers to other regions of the genome such as the beta cinnamomin elicitin gene gave very low levels of sensitivity compared to the ITS primers. In comparison with DNA detection we found that the limit of detection using baiting was 4 mcg mycelium g soil-1. Results below this limit were unreliable. The method suffered from the additional disadvantage that it took a long time in comparison to DNA detection. DNA detection methods do not distinguish between living and dead organisms in the soil. However it can be hypothesised that DNA is unlikely to persist for any significant length of time in soil. To test this, we added plasmid DNA to soil and tested the persistence of this DNA using a variety of methods such as precipitation of labelled DNA, southern blotting and PCR amplification. It was found that in general, in soils from different ecosystems, the bulk of the DNA was undetectable after 24 hours. The rate of DNA breakdown differed with the soil type. In some soils, the added DNA was not detected even after 2 hours, whereas in others it could be observed after 10 hours. The detection depended on the method. Southern blotting showed that although DNA could be observed at 10 hours, by 24 hours it was completely degraded. In contrast a PCR product could be obtained from the soil extracts up to 24 hours. In a separate experiment, plasmid DNA was detectable over a 24 hour incubation period in 5 soil samples from 5 different sites. The results suggest that DNA is degraded rapidly in soil and is unlikely to persist longer than 24 hours. The results in this thesis demonstrate that MALDI-TOF MS is a suitable alternative to agarose gel electrophoresis for analysis of PCR products. The technique is rapid, differentiates species from mixtures, is high-throughput and amenable to automation. Implementation will require further research to automate the primer extension assay to reduce the sensitivity to impurities in the DNA and to design parameters for sampling asymptomatic material.

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20

Mériaux, Céline. "Imagerie du système nerveux central par spectrométrie de masse MALDI." Thesis, Lille 1, 2011. http://www.theses.fr/2011LIL10059/document.

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Ces dernières années, l’imagerie par spectrométrie de masse MALDI s’est révélée être un outil puissant pour la recherche de biomarqueurs puisqu’elle permet d’effectuer l’analyse d’un large panel de composés endogènes et exogènes dans des coupes de tissu. Des développements restent néanmoins à faire pour l’amélioration de la détection des molécules. La préparation de l’échantillon, incluant les traitements chimiques et le dépôt de la matrice, est dépendante du tissu et des molécules d’intérêt et influence la qualité des spectres et des images. D’autre part, les outils bioinformatiques tels que les analyses multi variées apportent des informations sur les marqueurs en fonction des phénotypes. Ces étapes sont donc cruciales pour les applications de l’imagerie dans le domaine de la biologie. Tout d’abord, nous nous sommes donc axés sur le développement de nouvelles matrices adaptées à l’imagerie MALDI telles que les matrices ioniques. Ensuite, ces développements ont été appliqués au modèle invertébré sangsue médicinale, aux stades embryonnaires et adultes, afin de comparer les mécanismes biologiques intervenant lors de l’édification du système nerveux central et de la régénération nerveuse après lésion de ce système. Enfin, des études sur des atteintes neurologiques ont été entreprises afin de comprendre les facteurs clés impliqués dans la balance régénération/dégénérescence. Ainsi, les études des échantillons d’hippocampes humains ont révélés l’existence de protéines associées à une distribution particulière correspondant à des couches des neurones anormalement présents dans l’hippocampe des patients épileptiques
In recent years, MALDI mass spectrometric imaging has proved to be a powerful tool for biomarker research. This technology allows the analysis of a wide range of endogenous and exogenous compounds in tissue sections. Many developments need to be undertaken to improve the detection of molecules. The sample preparation, including chemical treatment and deposition of the matrix, is dependent on the tissue and molecules of interest and influences the quality of spectra and images. In addition, the bioinformatics tools such as multivariate analysis provide informations on the markers according to phenotypes. These steps are crucial for imaging applications in the field of biology. First of all, we focused on the development of new matrices suitable for MALDI imaging such as ionic matrices. Secondly, these developments have been applied to the invertebrate model, the medicinal leech, at embryonic and adult stages, to compare the biological mechanisms involved in the establishment of the central nervous system and nerve regeneration after injury of this system. Finally, studies of neurological damage have been undertaken to understand the key factors involved in the balance regeneration/degeneration. Thus, studies of human hippocampi samples have revealed the existence of proteins associated with a particular distribution corresponding to layers of neurons abnormally present in the hippocampus of epileptic patients

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21

Luizete, Milena Fontes. "Aplicações de MALDI-MS na análise de peptídeos produzidos por cianobactérias /." Araraquara, 2017. http://hdl.handle.net/11449/151215.

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Orientador: Humberto Márcio Santos Milagre
Banca: Saulo Santesso Garrido
Banca: Dulce Helena Siqueira Silva
Banca: Moacir Rossi Forin
Banca: João Henrique Ghilardi Lago
Resumo: Neste trabalho, a técnica Matrix Assisted Laser Dersoption/Ionization - Mass Spectrometry (MALDI-MS) foi utilizada em diferentes aplicações para a análise de peptídeos produzidos por cianobactérias. Esta técnica é extremamente rápida, possui alta resolução, requer pouquíssimo preparo de amostra e não permite que possíveis contaminantes presentes na amostra interfiram na análise. Por estes motivos MALDI- MS tem sido amplamente utilizada não somente na detecção de diferentes variantes de cianopeptídeos, mas também para quantificação das cianotoxinas. Este trabalho utilizou um sistema binário de matriz para a quantificação de microcistinas, utilizando as matrizes ácido α-ciano-4-hidroxicinamico e o ácido sinapínico (50/50, v/v) para obter uma mistura homogênea de matriz/analito, superando a baixa reprodutibilidade inerente da técnica MALDI-MS. Paralelamente, foi realizado o imageamento de espécies de cianobactérias de água doce cultivadas em meio sólido por MALDI-TOF-MS. Os resultados obtidos apresentaram novas perspectivas com relação à distribuição espacial dos peptídeos produzidos pelas espécies de cianobactérias estudadas, como a nodularina-R (m/z 825) e nodularina-[Har] (m/z 839) produzida pela espécie Nodularia harveyana PCC 7804, os peptídeos MC-LR (m/z 995) produzidos pela espécie Microcystis aeruginosa PCC 7820, e o sideróforo anaquelina (m/z 761.3) produzidos pela espécie Anabaena Cylindrica PCC 7122. Além disto, amostras da floração de cianobactérias, que ocorre na ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In this work, the technique Matrix Assisted Laser Dersoption/Ionization - Mass Spectrometry (MALDI-MS) were used in different application for analysis of peptides produces by cyanobacteria. This technique is extremely fast, has high resolution, requires very little sample preparation and does not allow any contaminants present in the sample to interfere with the analysis. For these reasons, MALDI-MS has been widely used not only in the detection of different variants of cyanopeptides, but also for the quantification of cyanotoxins. This work utilized a matrix binary system for the quantification of microcystins, using the α-cyano-4-hydroxynamic matrix and sinapinic acid (50/20, v/v) to obtain a homogeneous matrix/analyte mixture, overcoming the low reproducibility inherent to the MALDI-MS technique. Simultaneously, we performed the imaging of freshwater cyanobacteria cultivated in solid medium by MALDI-TOF-MS. The results obtained presented new perspectives regarding the spatial distribution of the peptides produced by the studied cyanobacteria species, for exemple the nodularin-R (m/z 825), nodularin-[Har] (m/z 839) for the species Nodularia harveyana PCC 7804, MC-LR peptides (m/z 995) for the species Microcystis aeruginosa PCC 7820, and the siderophore anaquelin (m/z 761.3) for the species Anabaena cylindrica PCC 7122. In addition, samples of cianobacteria's bloom, present in Salto Grande Lagoon, located in Americana-SP, were analyzed and were identified some peptides, being four microcystins (MC-YR, MC-YR, MC-Hil and MC-RR), four aeruginosins (602, 298 A, 644 and 968), two cyanopeptolins (972 and 986), and one variant of microviridin(1707) ... (Full abstract, click on electronic access below).
Doutor

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22

Lemaire, Rémi. "Nouveaux développements pour l'imagerie par spectrométrie de masse MALDI : applications aux problèmes biologiques et à la recherche de biomarqueurs dans le cancer de l'ovaire." Paris 6, 2006. http://www.theses.fr/2006PA066288.

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23

Robins, Chad LaJuan. "Nonpolar matrices for matrix assisted laser desportion ionization - time of flight - mass spectrometry." Cincinnati, Ohio : University of Cincinnati, 2005. http://www.ohiolink.edu/etd/view.cgi?acc%5Fnum=ucin1115293526.

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Thesis (Ph. D.)--University of Cincinnati, 2005.
Title from electronic thesis title page (viewed May 18, 2006). Includes abstract. Keywords: Matrix Assisted Laser Desorption/Ionization Mass Spectrometry; MALDI; Nonpolar Matrices; Charge-Transfer Ionization; Crude Oil; Mass Spectrometry. Includes bibliographical references.

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24

Crumpton, Jason. "Click Chemistry on DNA and Targeting RNA structure with Peptide Boronic Acids." Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/77076.

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The utilization of click chemistry to perform inter- and intramolecular ligation on DNA has become ubiquitous in the literature. Advances in copper (I) stabilizing ligands that prevent DNA degradation via redox pathways have provided nucleic acid researchers access to the efficiency and quantitative nature of the click reaction. The majority of ligation procedures in the literature are performed in solution after DNA assembly and modification with alkyne reporter groups. However, without specialty alkyne reagents that can be sequentially and selectively deprotected, the solution phase method requires that the click reaction be performed on all DNA-attached alkynes simultaneously. Therefore, the variability of the azide reagent is limited to a singular R group. However, performing the click reaction on DNA during synthetic elongation (immediately after each alkyne installation) allows for the possibility of performing multiple click reactions with variable azide reagents. Unfortunately, most solid phase click procedures require long reaction times or the utilization of microwave irradiation to accelerate the reaction. The development of methods for the ligation of azides to alkynes without the use of microwave irradiation on solid phase is potentially very useful. Herein, we report a simple, efficient, and robust solid phase synthetic method for the ligation of azido-diamondoids to the alkyne-modified phosphate backbone of DNA with click chemistry using [Cu(CH₃CN)₄]PF₆ without stabilizing ligand. Interestingly, it was found that as the size of diamondoid increased, a corresponding increase in melting temperature of hybridized duplexes was observed. The developed method has the potential to complement existing DNA ligation procedures for applications in biotechnology and diagnostics. Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. It is well known that RNA ligands incorporating basic and intercalating moieties display high RNA affinity. Unfortunately, these ligands are also often plagued by promiscuous binding to off-target substrates. Due to the potential utility of RNA ligands in biology and medicine, it is imperative to elucidate RNA binders which display high specificity as well as affinity. Boronic acid peptides promise unique RNA binding motifs through the interaction between the empty p-orbital of boron and the 2'-hydroxyl group of RNA. Herein, we describe the incorporation of lysine and phenylalanine boronic acid analogues into a branched peptide combinatorial library in an effort to impart increased selectivity towards the HIV-1 Rev Response Element (RRE). We were able to easily select and deconvolute 6 resulting "hit" peptides from 65,536 unique library members by high throughput screening and de novo sequencing. Although we were unable to evaluate peptide selectivity towards RRE due to general insolubility in aqueous media, we demonstrated the efficient deconvolution of a branched peptide library that incorporates boronic acids.
Ph. D.

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25

Crumpton, Jason B. "Click Chemistry on DNA and Targeting RNA structure with Peptide Boronic Acids." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/77076.

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Анотація:

The utilization of click chemistry to perform inter- and intramolecular ligation on DNA has become ubiquitous in the literature. Advances in copper (I) stabilizing ligands that prevent DNA degradation via redox pathways have provided nucleic acid researchers access to the efficiency and quantitative nature of the click reaction. The majority of ligation procedures in the literature are performed in solution after DNA assembly and modification with alkyne reporter groups. However, without specialty alkyne reagents that can be sequentially and selectively deprotected, the solution phase method requires that the click reaction be performed on all DNA-attached alkynes simultaneously. Therefore, the variability of the azide reagent is limited to a singular R group. However, performing the click reaction on DNA during synthetic elongation (immediately after each alkyne installation) allows for the possibility of performing multiple click reactions with variable azide reagents. Unfortunately, most solid phase click procedures require long reaction times or the utilization of microwave irradiation to accelerate the reaction. The development of methods for the ligation of azides to alkynes without the use of microwave irradiation on solid phase is potentially very useful. Herein, we report a simple, efficient, and robust solid phase synthetic method for the ligation of azido-diamondoids to the alkyne-modified phosphate backbone of DNA with click chemistry using [Cu(CH₃CN)₄]PF₆ without stabilizing ligand. Interestingly, it was found that as the size of diamondoid increased, a corresponding increase in melting temperature of hybridized duplexes was observed. The developed method has the potential to complement existing DNA ligation procedures for applications in biotechnology and diagnostics. Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. It is well known that RNA ligands incorporating basic and intercalating moieties display high RNA affinity. Unfortunately, these ligands are also often plagued by promiscuous binding to off-target substrates. Due to the potential utility of RNA ligands in biology and medicine, it is imperative to elucidate RNA binders which display high specificity as well as affinity. Boronic acid peptides promise unique RNA binding motifs through the interaction between the empty p-orbital of boron and the 2'-hydroxyl group of RNA. Herein, we describe the incorporation of lysine and phenylalanine boronic acid analogues into a branched peptide combinatorial library in an effort to impart increased selectivity towards the HIV-1 Rev Response Element (RRE). We were able to easily select and deconvolute 6 resulting "hit" peptides from 65,536 unique library members by high throughput screening and de novo sequencing. Although we were unable to evaluate peptide selectivity towards RRE due to general insolubility in aqueous media, we demonstrated the efficient deconvolution of a branched peptide library that incorporates boronic acids.
Ph. D.

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26

Walton, Barbara Lynn. "A Study of Silver: an Alternative Maldi Matrix for Low Weight Compounds and Mass Spectrometry Imaging." Thesis, University of North Texas, 2014. https://digital.library.unt.edu/ark:/67531/metadc499981/.

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Soft-landing ion mobility has applicability in a variety of areas. The ability to produce material and collect a sufficient amount for further analysis and applications is the key goal of this technique. Soft-landing ion mobility has provided a way to deposit material in a controllable fashion, and can be tailored to specific applications. Changing the conditions at which soft-landing ion mobility occurs effects the characteristics of the resulting particles (size, distribution/coverage on the surface). Longer deposition times generated more material on the surface; however, higher pressures increased material loss due to diffusion. Larger particles were landed when using higher pressures, and increased laser energy at ablation. The utilization of this technique for the deposition of silver clusters has provided a solvent free matrix application technique for MALDI-MS. The low kinetic energy of incident ions along with the solvent free nature of soft-landing ion mobility lead to a technique capable of imaging sensitive samples and low mass analysis. The lack of significant interference as seen by traditional organic matrices is avoided with the use of metallic particles, providing a major enhancement in the ability to analyze low mass compounds by MALDI.

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27

Lu, Kuan. "Optimization Of Sublimation Conditions for Surface Layer Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry Imaging (SL-MALDI- Tof MSI) of Polymer Surfaces." University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron1524846943404769.

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28

Jacksén, Johan. "Improved techniques for CE and MALDI-MS including microfluidic hyphenations foranalysis of biomolecules." Doctoral thesis, KTH, Analytisk kemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-27342.

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In this thesis, improved techniques for biomolecule analysis using capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and hyphenations between those have been presented.A pre-concentration method which is possible to apply in both techniques, has also been investigated. In this work the off-line MS mode has been used either in the form of fractionation (Paper I) or by incorporating the MALDI target in the CE separation system (Paper II).In Paper I, a protocol for CE-MALDI analysis of cyanogen bromide digested bacteriorhodopsin (BR) peptides as model integral membrane protein peptides were established. Also, an improved protocol for partially automated manufacturing of a concentration MALDI-target plate is presented. The design of the targets was suitable for the fractions from the CE. A novel technique for the integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a micro canal functioning as a MALDI target window. A protein separation was obtained and detected with MALDI-MS analysis in the micro canal. A method has been developed for detection of monosaccharides originating from hydrolysis of a single wood fiber performed in a micro channel, with an incorporated electromigration pre-concentration step preceding CE analysis in Paper III. The pre-concentration showed to be highly complex due to the fact that several parameters are included that affecting each other. In Paper IV a protocol using enzymatic digestion, MALDI-TOF-MS and CE with laser induced fluorescence (LIF) detection for the investigation of the degree of substitution of fluorescein isothiocyanate (FITC) to bovine serum albumin (BSA), as a contact allergen model system for protein-hapten binding in the skin, is presented. The intention of a further CE-MALDI hyphenation has been considered during the work. In Paper V 2,6-dihydroxyacetophenone (DHAP) was investigated, showing promising MALDI-MS matrix properties for hydrophobic proteins and peptides. 2,5-dihydroxybenzoic acid (DHB) was undoubtedly the better matrix for the hydrophilic proteins, but its performance for the larger and hydrophobic peptides was not optimal. Consequently, DHAP can be used as a compliment matrix for improved analysis of hydrophobic analytes.
QC 20101214

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29

Sorensen, Christina M. "ESI-MS and MALDI-TOF-MS for the characterization and analysis of metallo-oligomers and proteins." Laramie, Wyo. : University of Wyoming, 2005. http://proquest.umi.com/pqdweb?did=1031044031&sid=4&Fmt=2&clientId=18949&RQT=309&VName=PQD.

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30

Hoteling, Andrew J. "MALD/I TOF PSD and CID : understanding precision, resolution, and mass accuracy and MALD/I TOFMS : investigation of discrimination issues related to solubility /." Philadelphia, Pa. : Drexel University, 2004. http://dspace.library.drexel.edu/handle/1860/318.

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31

Júnior, João Nobrega de Almeida. "Padronização da espectrometria de massa MALDI-TOF para identificação de cepas de Trichosporon spp. de importância médica." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5144/tde-05052014-110905/.

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O gênero Trichosporon é composto por leveduras artrosporadas do Filo Basidiomycota e é conhecido agente de infecção fúngica invasiva (IFI) em pacientes imunodeprimidos ou com outros fatores de risco. Em pacientes onco-hematológicos é a principal levedura responsável por IFI depois do gênero Candida. Entre as espécies responsáveis por infecções no homem encontram-se: T. asahii, T. inkin, T. mucoides, T. dermatis, T. jirovecii, T. ovoides, T. cutaneum, T. montevideense, T. domesticum, T. asteroides, T. coremiiforme, T. faecale, T. dohaense, T. lactis, T. japonicum. A tecnologia de identificação de fungos por espectrometria de massa (SM) MALDI-TOF ainda carece de padronização para identificação de fungos do gênero Trichosporon, mas a literatura mostra resultados encorajadores. O objetivo deste estudo é padronizar a técnica de espectrometria de massa MALDI-TOF para a identificação das espécies do gênero Trichosporon de importância médica. O estudo foi realizado em cooperação entre a Divisão de Laboratório Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (DLC, HC-FMUSP), Instituto de Medicina Tropical da USP (IMT-USP), Instituto Adolfo Lutz (IAL) e Laboratoire de Parasitologie-Mycologie do Hospital Saint Antoine de Paris, vinculado ao grupo de pesquisa INSERM/UPMC UMR S945 \"Immunité et Infection\" Faculté de Medecine et Université Pierre et Marie Curie de Paris. Noventa e três cepas/isolados foram analisado(a)s, sendo dezenove cepas de referência adquiridas junto à coleção holandesa Centraalbureau Schimmelcultures (CBS), 19 isolados do HC-FMUSP e IAL, e 55 isolados de diferentes hospitais franceses. A identificação molecular foi realizada através do sequenciamento da região IGS1 do rDNA e foi considerada como método de referência. O protocolo de extração de proteínas foi estabelecido através da comparação do desempenho de três metodologias (Bruker®, Cassagne et al., Sendid et al.). Os espectros de massa foram obtidos no laboratório de bacteriologia do Hospital Saint Antoine de Paris através do aparelho Microflex LT®. A interpretação dos resultados qualitativos e quantitativos (logscore) foi realizada através do Software Biotyper 3.0®. O desempenho de identificação do banco de espectros de referência Biotyper 3.0® foi comparado a outros cinco bancos criados a partir de espectros de referência (ERs) derivados de 18 cepas de referência CBS, sete isolados clínicos e 11 ERs do banco Biotyper 3.0. O protocolo de extração de proteínas descrito por Sendid et al. foi escolhido como protocolo de referência pois os espectros produzidos tiveram logscore superiores àqueles obtidos através do método do fabricante. O banco de ERs Biotyper 3.0® apresentou 32,3% de identificações corretas das espécies, sendo que o banco de ERs in house (número 5, constituído cepas CBS e isolados clínicos) apresentou 98,5% de identificações de espécies. Espectros de referência do banco de dados Biotyper 3.0® foram submetidos à identificação com a utilização dos ERs criados a partir de cepas CBS e isolados clínicos e foram evidenciados com erros de identificação: T. mucoides (2), T. ovoides (1) e T. cutaneum (2). Após padronização do protocolo de extração e criação de banco de ERs com cepas CBS e isolados clínicos caracterizados pelo sequenciamento da região IGS, a SM por MALDI-TOF apresentou-se como potente uma ferramenta para a identificação de fungos do gênero Trichosporon. O banco de ERs Biotyper 3.0® apresentou um fraco desempenho, relacionado a ERs que foram criados a partir de cepas mal identificadas
Trichosporon spp. are arthrospored yeasts from the Filum Basidiomycota that are known to produce invasive fungal infection (IFI) in patients with immunosupression or other risk factors. After Candida, Trichosporon is the second genus of yeasts responsible for IFI in patients with onco-hematological diseases. The most important species related to human infection are: T. asahii, T. inkin, T. mucoides, T. dermatis, T. jirovecii, T. ovoides, T. cutaneum, T. montevideense, T. domesticum, T. asteroides, T. coremiiforme, T. faecale, T. dohaense, T. lactis, T. japonicum. The technology of mass spectrometry (MS) for identification of Trichosporon species has not yet been standardized. However, preliminary promising results can be found in the literature. The objective of this study is to analyse and validate MS MALDI-TOF for the identification of Trichosporon species of medical relevance. This was a multicentric study with collaboration from the Central Laboratory Section from Clinics Hospital of the Medical School from the University of São Paulo (DLC-HCFMUSP), Tropical Medicine Institute from the University of São Paulo (IMT-USP), Instituto Adolfo Lutz (IAL) and Laboratoire de Parasitologie-Mycologie from the Hospital Saint Antoine of Paris and INSERM/UPMC UMR S945 \"Immunité et Infection\", Faculté de Medecine et Université Pierre et Marie Curie of Paris. Ninety three strains/isolates belonging to sixteen Trichosporon species were analysed. Nineteen were purchased from Centraalbureau Schimmelcultures (CBS) yeast collection, 19 belonged to HC-FMUSP and IAL collections, 55 belonged to different French collections. The reference identification method was the IGS1 rDNA sequencing. A protein extraction protocol was first established after comparing the performance of three different methodologies (Bruker(TM), Cassagne et al., Sendid et al.). The mass spectra were obtained through a Microflex LT(TM) mass spectrometer located at the bacteriology laboratory from Saint Antoine Hospital, Paris. Mass spectra, qualitative and quantitative results were produced through the software Biotyper 3.0(TM). The performance of the original main spectrum (MSP) library was compared to other 5 in house libraries built with the combination of MSPs derived from CBS strains (18), clinical strains (7) or (Bruker Daltonics/BD, Germany/USA) (11). The extraction protocol described by Sendid et al. showed better performance when compared to the manufacturer\'s one and was chosen for the subsequent extractions. Among the 6 different reference spectra databases tested, a specific one composed of 18 reference strains plus 7 clinical isolates (database 5) allowed the correct identification of 66 amongst 67 clinical isolates (98,5%). Biotyper 3.0 library produced only 32,3% of correct identifications. Biotyper\'s MSPs were submitted to cross-identification with MSPs derived from CBS strains and clinical isolates and misidentified original MSPs were identified: T. mucoides (2), T. ovoides (1) e T. cutaneum (2). While until now less widely applied to basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level. The MSP library Biotyper 3.0 showed a poorer performance which was due to misidentified strains utilized as reference for the MSPs

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32

Holcomb, April M. Owens Kevin G. "Investigation into the ionization mechanism occurring in matrix assisted laser desorption ionization and factors affecting ion flight time in MALDI time-of-flight mass spectrometry /." Philadelphia, Pa. : Drexel University, 2009. http://hdl.handle.net/1860/3161.

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33

Åminne, Ann. "Evaluation of preanalytic methods in order to shorten the processing time before identification of fungal microorganisms by the MALDI-TOF MS." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-255132.

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Identification of fungi is based on macroscopic observations of morphology and microscopic characteristics. These conventional methods are time-consuming and requires expert knowledge. For the past years Matrix-assisted laser desorption ionization-time of flight mass spectrometry has been used for routine bacterial identification in clinical laboratories but not yet in the same extension for fungi. In this study three preanalytic preparation methods for fungi were evaluated in order to shorten the processing time in routine laboratory performance. Clinically relevant strains (n=18) of molds and dermatophytes were cultivated on agar plates and prepared according to the different preparation methods for protein extraction. Each strain was analyzed in quadruplicate by the MALDI Biotyper and the database Filamentous Fungi Library 1.0. The results showed that the genus and species identification rates of the least time-consuming direct extraction method were 33% and 11% respectively. Using the formic acid extraction method, the genus and species identification rates were 83% and 44%, respectively. For the longest sample preparation method, liquid media culturing before formic acid extraction, successfully identified all strains except one, which resulted in an identification rate of 94% and 78% respectively. This study shows that preparing samples in cultured liquid media MADLI-TOF MS effectively identified fungal strains to both genus- and species-level. This method was however too time-consuming and cumbersome to be recommended as a replacement to the conventional method. Future studies should be aimed at expanding the reference library and making the direct extraction method more reproducible in terms of obtaining more reliable identification rates.

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34

Andrade, Laíse de Oliveira. "Métodos rápidos para identificação microbiana aplicados ao monitoramento ambiental de salas limpas: ênfase na tecnologia MALDI-TOF." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/9/9139/tde-26102017-114152/.

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A espectrometria de massas baseada na tecnologia MALDI-TOF (do inglês, matrix-assisted laser desorption ionization-time of flight) (MALDI-TOF MS) tem sido cada vez mais incorporada à rotina de identificações microbiológicas nos laboratórios farmacêuticos de controle de qualidade, principalmente para as atividades do Programa de Monitoramento Ambiental de Salas Limpas. Isso porque o longo tempo necessário para a obtenção dos resultados por meio de métodos convencionais tem incentivado a procura por técnicas que permitam métodos rápidos. O objetivo deste trabalho foi avaliar a adequação da técnica MALDI-TOF MS para a identificação de bactérias isoladas do ambiente de salas limpas utilizadas em algumas etapas da produção de uma vacina viral. Treze espécies bacterianas conhecidas, normalmente isoladas das salas limpas estudadas, e cinco cepas ATCC foram identificadas pela técnica MALDI-TOF MS e por uma técnica bioquímica (BBL Crystal®). O desempenho da técnica MALDI-TOF MS foi superior ao da técnica bioquímica na identificação correta das espécies bacterianas (88,89% e 38,89%, respectivamente) e produziu menos identificações não confiáveis (5,55% e 22,22%, respectivamente). Os resultados evidenciaram que a técnica MALDI-TOF MS pode ser implementada para identificação rotineira de bactérias em um laboratório de controle de qualidade farmacêutico. Entretanto, a dependência de bases de dados exige estudos adicionais de isolados não identificados e, se apropriado, a adição destes a uma base de dados interna. O aperfeiçoamento de métodos de identificação microbiana é muito relevante no contexto de salas limpas, pois permitem ações corretivas e proativas essenciais para garantir a segurança microbiológica do processamento asséptico.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been increasingly introduced in routine microbiological identifications of pharmaceutical quality control laboratories, mainly for the activities of the Environmental Monitoring Program of Clean Rooms. The long time needed to obtain the results through conventional methods has stimulated the search for techniques that allow rapid methods, as MALDI-TOF MS. Thus, the objective of this work was to evaluate the suitability of the MALDI-TOF MS technique for the identification of bacteria isolated from the environment of clean rooms used in some stages of the production of a viral vaccine. Thirteen bacterial species commonly isolated from clean rooms studied and five strains ATCC were identified by MALDI-TOF MS technique and by a biochemical technique (BBL Crystal® System). Performance of MALDI-TOF MS was better than biochemical technique for correct species identifications (88.89% and 38.89%, respectively) and produced fewer unreliable identifications (5.55% and 22.22%, respectively). MALDI-TOF MS can be implemented for routine identification of bacteria in a pharmaceutical quality control laboratory. However, as a database-dependent system, maybe some isolated not identified by this technique must be additionally studied and, if appropriate, added to an in-house database.

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35

Gibb, Sebastian. "Entwicklung einer flexiblen bioinformatischen Plattform zur Analyse von Massenspektrometriedaten." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-178678.

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Sowohl in der Klinischen Labormedizin, der Klinischen Mikrobiologie als auch in der Pathologie ist die Massenspektrometrie (MS) ein bedeutender Bestandteil der Diagnostik geworden. Der Fortschritt in der Gerätetechnik ermöglicht in kurzer Zeit viele, hochaufgelöste Spektren zu generieren. Diese Informationsvielfalt macht die manuelle Auswertung durch den Anwender sehr kompliziert bis unmöglich. Aus diesem Grund ist die Unterstützung durch bioinformatische Programme notwendig. Für die Reproduzierbarkeit der Ergebnisse und die Qualitätskontrolle ist es essentiell, dass die verwendeten Algorithmen transparent und die Programme als Open Source Software (OSS) frei verfügbar sind (Aebersold and Mann, 2003). Das Ziel dieser Arbeit war die Entwicklung von MALDIquant, einer unter der GNU General Public License (GPL) stehenden, flexiblen OSS, die für die o.g. Anwendungsbereiche modernste Algorithmen für die komplette Analyse bietet und in der freien Programmiersprache R (R Core Team, 2014) geschrieben ist. Im Zusammenspiel mit dem dazugehörigen Paket MALDIquantForeign ist MALDIquant in der Lage die üblichen Dateiformate der verschiedenen MS-Geräte zu verarbeiten. Dadurch ist MALDIquant hersteller- und geräteunabhängig und eignet sich nicht nur für MALDI/TOF, sondern für alle zweidimensionalen MS-Daten. Angefangen vom Datenimport über die Prozessierung bis hin zur Analyse der Spektren bietet MALDIquant eine komplette Analyse-Pipeline und implementiert state-of-the-art Methoden. Neben weit verbreiteten Verfahren zur Baseline Correction und Peak Detection zeichnet sich MALDIquant besonders durch ein hervorragendes Peak Alignment aus. Dieses ist sehr genau und aufgrund des Fokus auf die Peaks schneller als die meisten anderen Verfahren und weitestgehend unabhängig von der Qualität der Intensitätenkalibrierung. Eine weitere Stärke von MALDIquant ist die Möglichkeit, eigene Algorithmen zu integrieren, sowie den Ablauf der Analyse den individuellen Bedürfnissen anzupassen. In der beispielhaften Analyse der Daten von Fiedler et al. (2009) konnten durch MALDIquant Peaks gefunden werden, die Patienten mit Pankreaskarzinom von nicht erkrankten Probanden unterscheiden. Einige dieser Peaks wurden bereits in anderen Publikationen beschrieben. Neben diesem Beispiel hat MALDIquant seine Nützlichkeit bereits in verschiedenen Anwendungsbereichen und Publikationen bewiesen, wie etwa in Ouedraogo et al. (2013) oder Jung et al. (2014).

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36

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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37

Watson, R. Craig Jr. "Laser-Ionization Time-of-Flight Mass Spectrometry of High Molecular Mass Inorganic Complexes." Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/35554.

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Анотація:

Laser-Ionization Time-of-Flight Mass Spectrometry (LI-TOF-MS) is a sophisticated tool for the molecular-weight determination and structural characterization of a variety of molecules. Advances in instrumentation and ionization methods have recently expanded its role in the analysis of high-mass analytes. Large multimetallic complexes, which are efficient solar-energy converters, rely heavily on their chemical structure for optimum operation. Molecular mass determinations of these multimetallic complexes have been problematic due to their lability and high molecular weights.

This thesis describes the characterization of a LI-TOF-MS instrument and confirmation of theoretical time-of-flight mass-separation principles. Several test cases demonstrate the instrument's proper operation and calibration for a wide mass range of analytes. Mass spectral results of three organometallic compounds: i. [Ir(dpp)₂Cl₂](PF₆), ii. {[(bpy)₂Ru(dpp)]₂IrCl₂}(PF₆)₅, and iii. {[(bpy)₂Ru(dpp)]₂RuCl₂}(PF₆)₅ under a variety of laser ionization and sample preparation conditions are compared. A complete structural characterization of the monometallic complex, [Ir(dpp)₂Cl₂](PF₆), is presented. The two trimetallic analytes fragmented easily, but significant components of the molecules are successfully identified. After optimizing the ionization and analytical procedure, LI-TOF-MS proved useful in the analysis of high molecular mass metal complexes.
Master of Science

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38

Saleem, Saira. "Proteomic profiling of pro and active matrix metalloproteinases using tandem mass spectrometry. optimization of affinity chromatography and nHPLC-MALDI-MS/MS for proteomic discrimination of matrix metalloproteinases in pre-clinical cancer model." Thesis, University of Bradford, 2012. http://hdl.handle.net/10454/5752.

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Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.

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39

Jung, Seokwon. "Surface characterization of biomass by imaging mass spectrometry." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/45906.

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Lignocellulosic biomass (e.g., non food-based agricultural resides and forestry wastes) has recently been promoted for use as a source of bioethanol instead of food-based materials (e.g., corn and sugar cane), however to fully realize these benefits an improved understanding of lignocellulosic recalcitrance must be developed. The primary goal of this thesis is to gain fundamental knowledge about the surface of the plant cell wall, which is to be integrated into understanding biomass recalcitrance. Imaging mass spectrometry by TOF-SIMS and MALDI-IMS is applied to understand detailed spatial and lateral changes of major components in the surface of biomass under submicron scale. Using TOF-SIMS analysis, we have demonstrated a dilute acid pretreated poplar stem represented chemical differences between surface and bulk compositions. Especially, abundance of xylan was observed on the surface while sugar profile data showed most xylan (ca. 90%) removed from the bulk composition. Water only flowthrough pretreated poplar also represented difference chemistry between surface and bulk, which more cellulose revealed on the surface compared to bulk composition. In order to gain the spatial chemical distribution of biomass, 3-dimensional (3D) analysis of biomass using TOF-SIMS has been firstly introduced in the specific application of understanding recalcitrance. MALDI-IMS was also applied to visualize different molecular weight (e.g., DP) of cellulose oligomers on the surface of biomass.

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40

Kuismin, M. (Markku). "On regularized estimation methods for precision and covariance matrix and statistical network inference." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526220802.

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Abstract Estimation of the covariance matrix is an important problem in statistics in general because the covariance matrix is an essential part of principal component analysis, statistical pattern recognition, multivariate regression and network exploration, just to mention but a few applications. Penalized likelihood methods are used when standard estimates cannot be computed. This is a common case when the number of explanatory variables is much larger compared to the sample size (high-dimensional case). An alternative ridge-type estimator for the precision matrix estimation is introduced in Article I. This estimate is derived using a penalized likelihood estimation method. Undirected networks, which are connected to penalized covariance and precision matrix estimation and some applications related to networks are also explored in this dissertation. In Article II novel statistical methods are used to infer population networks from discrete measurements of genetic data. More precisely, Least Absolute Shrinkage and Selection Operator, LASSO for short, is applied in neighborhood selection. This inferred network is used for more detailed inference of population structures. We illustrate how community detection can be a promising tool in population structure and admixture exploration of genetic data. In addition, in Article IV it is shown how the precision matrix estimator introduced in Article I can be used in graphical model selection via a multiple hypothesis testing procedure. Article III in this dissertation contains a review of current tools for practical graphical model selection and precision/covariance matrix estimation. The other three publications have detailed descriptions of the fundamental computational and mathematical results which create a basis for the methods presented in these articles. Each publication contains a collection of practical research questions where the novel methods can be applied. We hope that these applications will help readers to better understand the possible applications of the methods presented in this dissertation
Tiivistelmä Kovarianssimatriisin estimointi on yleisesti ottaen tärkeä tilastotieteen ongelma, koska kovarianssimatriisi on oleellinen osa pääkomponenttianalyysia, tilastollista hahmontunnistusta, monimuuttujaregressiota ja verkkojen tutkimista, vain muutamia sovellutuksia mainitakseni. Sakotettuja suurimman uskottavuuden menetelmiä käytetään sellaisissa tilanteissa, joissa tavanomaisia estimaatteja ei voida laskea. Tämä on tyypillistä tilanteessa, jossa selittävien muuttujien lukumäärä on hyvin suuri verrattuna otoskokoon (englanninkielisessä kirjallisuudessa tämä tunnetaan nimellä ”high dimensional case”). Ensimmäisessä artikkelissa esitellään vaihtoehtoinen harjanne (ridge)-tyyppinen estimaattori tarkkuusmatriisin estimointiin. Tämä estimaatti on johdettu käyttäen sakotettua suurimman uskottavuuden estimointimenetelmää. Tässä väitöskirjassa käsitellään myös suuntaamattomia verkkoja, jotka liittyvät läheisesti sakotettuun kovarianssi- ja tarkkuusmatriisin estimointiin, sekä joitakin verkkoihin liittyviä sovelluksia. Toisessa artikkelissa käytetään uusia tilastotieteen menetelmiä populaatioverkon päättelyyn epäjatkuvista mittauksista. Tarkemmin sanottuna Lassoa (Least Absolute Shrinkage and Selection Operator) sovelletaan naapuruston valinnassa. Näin muodostettua verkkoa hyödynnetään tarkemmassa populaatiorakenteen tarkastelussa. Havainnollistamme, kuinka verkon kommuunien (communities) tunnistaminen saattaa olla lupaava tapa tutkia populaatiorakennetta ja populaation sekoittumista (admixture) geneettisestä datasta. Lisäksi neljännessä artikkelissa näytetään, kuinka ensimmäisessä artikkelissa esiteltyä tarkkuusmatriisin estimaattoria voidaan käyttää graafisessa mallinvalinnassa usean hypoteesin testauksen avulla. Tämän väitöskirjan kolmas artikkeli sisältää yleiskatsauksen tämänhetkisistä työkaluista, joiden avulla voidaan valita graafinen malli ja estimoida tarkkuus- sekä kovarianssimatriiseja. Muissa kolmessa julkaisussa on kuvailtu yksityiskohtaisesti olennaisia laskennallisista ja matemaattisista tuloksista, joihin artikkeleissa esitellyt estimointimenetelmät perustuvat. Jokaisessa julkaisussa on kokoelma käytännöllisiä tutkimuskysymyksiä, joihin voidaan soveltaa uusia estimointimenetelmiä. Toivomme, että nämä sovellukset auttavat lukijaa ymmärtämään paremmin tässä väitöskirjassa esiteltyjen menetelmien käyttömahdollisuuksia

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41

Borchardt, Lars, and Sven Grätz. "Mechanochemical polymerization – controlling a polycondensation reaction between a diamine and a dialdehyde in a ball mill." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-221833.

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The mechanochemical polycondensation between a diamine and a dialdehyde constitutes a sustainable alternative to classical solvent-based polymerization reactions. This process not only allows for a higher conversion and a shorter reaction time as compared to standard solvent-based syntheses of this conjugated polymer, but the reaction can also be adjusted by the energy introduced via the ball mill.

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42

Borchardt, Lars, and Sven Grätz. "Mechanochemical polymerization – controlling a polycondensation reaction between a diamine and a dialdehyde in a ball mill." Royal Society of Chemistry, 2016. https://tud.qucosa.de/id/qucosa%3A30231.

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Анотація:

The mechanochemical polycondensation between a diamine and a dialdehyde constitutes a sustainable alternative to classical solvent-based polymerization reactions. This process not only allows for a higher conversion and a shorter reaction time as compared to standard solvent-based syntheses of this conjugated polymer, but the reaction can also be adjusted by the energy introduced via the ball mill.

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43

Magalhães, Ilídio Miguel Teixeira. "Proteome of biofilm produced by a S. pseudintermedius strain." Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/14291.

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Mestrado em Bioquímica
Staphylococcus pseudintermedius is an opportunistic pathogenic bacterium responsible for most skin and post-surgical infections in dogs. The number of bacterial strains resistant to β-lactam antibiotics is increasing and are the major challenges now faced by veterinary medicine. Bacteria that produce biofilm are more resistant to treatment and thus, the production of this structure is already considered a virulence factor. In a biofilm, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) some of which are proteins. With the objective to know more of this array element, the characterization of the biofilm matrix proteome (BMP) from a highly virulent S. pseudintermedius strain isolated from a dog with severe pyoderma was performed. Biofilm was developed by culturing the S. pseudintermedius strain 5819/10 in specific media. The biofilm matrix was then be separated from bacterial cells and evaluated for their protein content and complexity. Finally, the proteome was separated by 1D electrophoresis and characterized by nanoLC-ESI-Q-TOF and analysed using bioinformatics tools. The BMP of strain S. pseudintermedius 5819/10 consisted in a diverse group of proteins, where 63% of the proteins could be related to either the extracellular region or the plasma membrane, as protein complexes, and most of them had functions essential to cell survival. However, it was not possible to establish a clear relation between them and biofilm formation. Proteins known to be involved in biofilm formation consisted mostly of regulator factors of biofilm formation as well as virulence factors of-mainly-bacterial cell adhesion and host colonization. The prevalence of adhesins and the almost total absence of proteins involved in EPS synthesis pointed to a biofilm matrix where cells are directly or indirectly closely glued together to each other.
Staphylococcus pseudintermedius (S.pseudintermedius) é uma bactéria patogénica oportunista, responsável pela maioria das infeções cutâneas e pós-cirúrgicas em cães. O número de estirpes resistentes a antibióticos β-lactâmicos está a aumentar constituindo actualmente um dos grandes desafios enfrentados pela medicina veterinária. As bactérias mais resistentes ao tratamento são aquelas que produzem biofilme sendo esta capacidade considerada um fator de virulência. Num biofilme, as bactérias estão envoltas numa matriz de substâncias poliméricas extracelulares (SPE), algumas das quais são proteínas. Tendo por objectivo obter mais informação acerca do biofilme, foi caracterizado o proteoma da matriz do biofilme de uma estirpe bastante virulenta de S. pseudintermedius isolada de um cão com piodermite profunda. Para tal cultivaram-se biofilmes da estirpe de S. pseudintermedius 5819/10 em meio apropriado, separou-se a matriz das suas células bacterianas e avaliou-se as proteínas presentes quanto ao seu conteúdo e complexidade. Posteriormente o proteoma foi separado por electroforese 1D, caracterizado por nanoLC-ESI-Q-TOF e analisado usando ferramentas bioinformáticas Constatou-se que o proteoma da matriz do biofilme da estirpe 5819/10 de S. pseudintermedius é muito diverso e que 63% das proteinas podem estar relacionadas com a região extracelular do biofilme ou da membrana plasmática na forma de complexos proteicos. Verificou-se também que a maioria das proteínas identificadas possui funções essenciais para a sobrevivência da bactéria mas não foi possível estabelecer uma relação clara entre elas e a formação de biofilmes. Algumas proteínas que se sabe estarem envolvidas na formação de biofilmes foram identificadas, tratam-se principalmente de factores reguladores da formação de biofilme e outros factores de virulência relacionados com a colonização de um hospedeiro a adesão bacteriana a uma superfície. A prevalência de adesinas e a ausência quase total de proteínas envolvidas na síntese de SPEs, forneceu dados que apoiam a hipótese que a matriz do biofilme do S. pseudintermedius 5819/10 seja constituída por células directamente ou indirectamente unidas entre si.

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44

Agatea, Lisa. "An integrated proteomic and genomic approach to study FAP patients without APC and MutHY mutations." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424509.

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Familial Adenomatous Polyposis (FAP) is one of the most important clinical forms of inherited susceptibility to colorectal cancer, that is characterized by the development of hundreds to thousands of adenomas in the colon and rectum during the second decade of life. FAP is due to a germline mutation in the APC gene or to biallelic variations of MutYH gene. Almost all patients will develop cancer if the disease is not identified and surgically treated at an early stage. The aim of this study was to characterize, by peptidomic and genetic approaches, 4 pa-tients that, although at the colonoscopy showed many polyps, they did not present any mutations of APC and MutYH genes (defined here unresolved FAP). Regarding the peptidomic study, MALDI-TOF analysis was performed on mutated and unresolved FAP patients. These data were compared with the one from adenoma patients, CRC patients and healthy control subjects. The peptide fingerprint of mutated FAP patients was obtained after performing statistical analysis. A subset of 45 ionic species was found differently expressed in the four groups considered, 12 of them peculiar of FAP patients. Four ionic species were found significantly different in the switch between adenoma and malignant carcinoma. In this study, the potentially prognostic peptides identified derive mainly from circulating proteins and some of them are involved in the inflammatory response. In particular, proteins such as Complement C3 and C4 are known to be cleaved by exoproteases that seem pathology-related. In the case of unresolved FAP patients, in order to better define a specific pattern, the data from MALDI-TOF were combined with whole exome sequencing. The peptidomics data clearly mark a substantial difference between mutated and unresolved FAP patients. Indeed, unresolved FAP patients have characteristics similar to the control subjects, adenoma patients, CRC patients but not to mutated FAP patients. To understand the possible molecular pathway involved in the unresolved FAP cases, the whole exome sequencing (WES) was performed. From WES data analysis, 285 genes present in all the four unresolved FAP patients were filtered and selected. Among them, the O-linked glycans pathway of the mucins was the most represented. In conclusion, in this study it was defined for the first time a specific panel of peptides for mutated FAP patients, that could be useful to monitor and predict the pathological evolution of adenocarcinoma malignancy. Furthermore, it was possible to characterize a preliminary genetic variations pattern for unresolved FAP patients, in which mucin genes might represent the key of the molecular pathway involved. However, further study are necessary to relate the identified mucin gene variations to their possible causative role in the polyposis. Future analysis of this pattern will be helpful, indeed, to better understand the interatome (the biological network that in-cludes the whole set of direct and indirect molecular interactions in a cell) of these un-resolved FAP patients.
La poliposi adenomatosa familiare (FAP) è una delle più importanti forme cliniche di cancro colo-rettale ereditario ed è caratterizzata dallo sviluppo di centinaia/migliaia di polipi adenomatosi nel colon e nel retto durante la seconda decade di vita. La FAP è causata da una mutazione germinale del gene APC o da varianti bialleliche del gene MutYH. Quasi tutti i pazienti FAP sviluppano il cancro se la patologia non viene precocemente identificata e trattata chirurgicamente. Lo scopo di questo lavoro è stato caratterizzare 4 pazienti in cui, nonostante l’esame colonscopico presentasse una poliposi conclamata, non risultavano mutazioni nei gene APC e MutYH (in questa tesi definiti pazienti FAP irrisolti) utilizzando un approccio integrato di peptidomica e genomica. Riguardo la peptidomica, il MALDI-TOF è stato utilizzato per studiare il profilo peptidico plasmatico di pazienti FAP mutati ed irrisolti comparando i dati ottenuti con quelli derivanti dallo studio di pazienti con adenoma, cancro colo-rettale e soggetti sani di controllo. Dopo analisi statistica è stato ottenuto il fingerprint peptidico dei pazienti FAP mutati. Sono state ottenute 45 specie ioniche differentemente espresse nei quattro gruppi considerati, 12 delle quali peculiari per i pazienti FAP. L’intensità di segnale di quattro di queste specie ioniche è stata trovata statisticamente alterata nello switch tra adenoma e carcinoma maligno. I peptidi potenzialmente prognostici identificati in questo studio derivano principalmente da proteine circolanti, alcune delle quali implicate nella risposta infiammatoria. In particolare è noto dalla letteratura che proteine del sistema del complemento come C3 e C4 vengono tagliate da esoproteasi che sembrano essere patologia correlate. Riguardo ai pazienti FAP irrisolti, per definirne un pattern specifico, i dati derivanti dall’analisi con il MALDI-TOF sono stati combinati con quelli ottenuti dal sequenzia-mento dell’esoma. I dati di peptidomica hanno chiaramente evidenziato le differenze tra pazienti FAP mutati e FAP irrisolti. Infatti i pazienti FAP irrisolti presentano caratteristiche simili a quelle dei soggetti di controllo, dei pazienti con adenoma e cancro colo rettale ma non a quelle dei pazienti FAP mutati. Allo scopo di capire la via di trasduzione del segnale implicata, è stato quindi eseguito il sequenziamento dell'esoma dei pazienti FAP irrisolti. Da questa analisi sono stati selezionati 285 geni variati in tutti i pazienti e tra questi la via di trasduzione del segnale della O-glicosilazione delle mucine è risultata la più rappresentata. In conclusione, in questo studio è stato definito per la prima volta un set peptidico specifico per i pazienti FAP mutati che potrebbe essere utilizzato per monitorare e predire l’evoluzione patologica della malattia. Inoltre è stato possibile caratterizzare un pattern preliminare per i pazienti FAP irrisolti in cui i geni delle mucine potrebbero rappresentare la chiave della via di trasduzione del segnale implicata. Ulteriori studi saranno necessari per correlare i geni delle mucine con la poliposi e costruire l'interatoma (network biologico definito come l’insieme di tutte le interazioni molecolari dirette e indirette che ci sono all'interno di una cellula e di un organismo) di questi pazienti FAP irrisolti.

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45

Ochoa, Mariela L. "Forensic and Proteomic Applications of Thermal Desorption Ion Mobility Spectrometry and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry." Ohio University / OhioLINK, 2005. http://www.ohiolink.edu/etd/view.cgi?ohiou1113585811.

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46

Macháčková, Petra. "Nové matrice pro MALDI-MS analýzu lipidů." Master's thesis, 2011. http://www.nusl.cz/ntk/nusl-298002.

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In this work focused on research of new matrices for MALDI-MS analysis of lipids several organic salts were tested as potential matrices for hydrocarbons and wax esters. It was shown previously that the effective ionization of lipids occurs in conjuction with a lithium cation to form adducts [M + Li] + . Therefore, lithium salts of aromatic acids were synthesized. Matrices were evaluated according to their MALDI properties and compared with lithium 2,5-dihydroxybenzoate, which is proven matrix for MALDI analysis of lipids. It was found that effectiveness of matrix depends on presence of hydroxyl group in matrix molecule. The best results were achieved with lithium vannilate, even in comparison with lithium 2,5-dihydroxybenzoate.

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47

Aiello, Donatella, Anna Napoli, Giovanni Sindona, and Bartolo Gabriele. "Protein characterization from natural matrices by maldi tof-tof." Thesis, 2014. http://hdl.handle.net/10955/443.

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48

Yang, Siao-Huei, and 楊筱蕙. "Concentration and Analysis of Peptides Using MALDI Ionic Liquid Matrices." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/93447220048370658723.

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碩士
國立東華大學
化學系
97
Sample preparation procedures such as desalting and concentration are key steps for a successful MALDI analysis of biological samples. A new approach of using ionic liquid matrix (ILM) α-cyano-4-hydroxycinnamate/3-aminoquinoline (CCA/3-AQ) as a solvent for the separation and preconcentration of peptides in aqueous samples prior to analysis by MALDI has been demonstrated. The samples were analyzed in-situ after the extraction without further sample transfer. Liquid liquid microextraction and single drop microextraction were employed for the extraction of peptides. MALDI analysis of protein digests in the presence of high concentration buffers and urea was investigated using CCA/3-AQ as a concentration solvent and a MALDI matrix.

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49

Li, Zong-Sian, and 李宗憲. "Qualitative Analysis of Peptides and Proteins by MALDI-TOF Mass Spectrometry Using Liquid Matrices." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/55156337709886526601.

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50

Krüger, Ralf [Verfasser]. "Untersuchungen zum Einbau von Analytionen in MALDI-Matrizes sowie zur Ionisation und Adduktbildung in der MALDI-Massenspektrometrie / vorgelegt von Ralf Krüger." 2003. http://d-nb.info/969681682/34.

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