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Lai-Rowcroft, Lindsay Ling Gi. "Novel surfaces for MALDI-MS." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/novel-surfaces-for-maldims(331dd97a-881e-4ed0-908f-d5947f3ebeba).html.
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Matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) for small molecule analysis has been plagued with inherent problems associated with matrix interference. The matrix plays an important role in MALDI-MS where it has the ability to absorb UV energy from the laser employed and transfer it to the analyte, acts as a proton donor and protecting the analytes from being obliterated. For decades, research has been performed to eradicate matrix interference by matrix avoidance, finding alternative matrices, suppression through sample preparation methods and via chemical modification.In this investigation a number of the above mentioned approaches have been undertaken. First, a mesoporous silica powder, SBA-16, functionalised with a phenyl group to absorb UV from the MALDI-MS laser gave unfruitful results due to inhomogeneous dispersion of the SBA-16 powder. Therefore the same material was prepared but as a thin film and a homogeneously coated surface was generated with the phenyl group incorporated into the silica and this was compared with a conventional matrix, 2,5-dihydroxybenzoic acid (DHB). This was by far the most sensitive method which was accurate, with little background noise and importantly for small molecule analysis clear of matrix interference. Other surface systems were also tested such as graphene on copper and silver on copper, but the functionalised SBA-16 thin film remained the best. Graphite and 2B pencil were also investigated for MALDI-MS but were compared with conventional matrices (DHB and α-cyano-4-hydroxycinnamic acid (CHCA)) in a functional genomics study. The ability of all methods to find subtle phenotypic differences in various yeast strains was assessed with the help of multivariate data analysis (MVDA). Although DHB came out best, 2B pencil produce notably good separations that correlated nicely with the different genotypes. Therefore in addition to conventional matrices, 2B pencil should be considered for functional genomic studies when MALDI-MS is used as it is such a rapid and inexpensive method. Finally, chemical modifications were performed on amino acids where picolinic acid was used to attach a chromophore to the compounds, therefore, allowing UV absorption from the laser. Upon attaching the picolinate UV absorbing group, the amino acid compounds were detected LC-MS at an increased intensity of 10 to 100-fold. Moreover, enhanced separation in LC-MS was also observed.This project has successfully investigated alternative approaches to matrix-free MALDI-MS analysis. Functionalised SBA-16 thin films were by far the best method and this novel surface for MALDI-MS has the potential to transform small molecule analysis.
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Leander, Ellinor. "Artidentifiering av mögelsvamp med MALDI-TOF MS." Thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-80166.
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Snabb och korrekt artidentifiering är avgörande för effektiv behandling av svampinfektioner, särskilt bland immunsupprimerade patienter. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) används rutinmässigt på kliniska laboratorier för identifiering av karaktäristiska proteinmönster hos bakterier och jästsvampar genom tolkning av proteinspektra i en masspektradatabas för korrekt artidentifiering. Mögelsvamparnas hårda cellvägg och heterogena växtsätt med varierande proteinuttryck beroende på mognadsstadie, försvårar identifiering med MALDI-TOF MS. Metodens tänkbara fördelar mot traditionella metoden mikroskopering är förkortade svarstider, säkrare artidentifiering av fler arter och mindre beroende av subjektiv morfologisk bedömning. Studiens syfte var att undersöka om MALDI-TOF MS kunde anpassas och användas för identifieringen av mögelsvamp i klinisk rutindiagnostik. Fyra referensstammar (Aspergillus niger, A. fumigatus, A.terreus, A.flavus) och ett kliniskt isolat (A.terreus) undersöktes. Preparationsmetoderna (I) fullständig myrsyraextraktion, (II) direktapplicering och (III) suspension i destillerat vatten användes för analys av sporer och frontmycel hos yngre och äldre mögelkulturer. Två olika masspektradatabaser för artidentifiering jämfördes; rutindatabasen BDAL och den specialiserade mögeldatabasen Filamentous Fungi Library. Även plocktekniken av mögelmaterial inför analys med MALDI-TOF MS utvärderades. Vid vissa tillfällen förbättrades artidentifieringen efter extraktion av mögelkulturerna, medan i andra fall var direktapplicering fullt tillräcklig. Mögelmaterial med mycket sporer tenderade ge något fler artidentifieringar i BDAL oavsett kulturernas ålder. Filamentous Fungi Library tenderade i vissa fall ge bättre resultat jämfört med BDAL för yngre kulturer. Fler studier krävs för att utvärdera och optimera MALDI-TOF MS som metod för artidentifiering av mögelsvamp.Rapid and accurate species identification is crucial for successful treatment of fungal infections, especially among immunosuppressed patients. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used routinely at clinical laboratories to identify characteristic protein patterns of bacteria and yeast by the interpretation of protein spectra in a database for accurate species identification. The hard cell wall of the mold and the heterogeneous growth with varying protein expression due to maturation, complicates identification with MALDI-TOF MS. The potential benefits of this method compared to microscopy as traditional method are shortened turn-around times, safer species identification of more species that is independent on subjective morphological assessment. The purpose of the study was to investigate whether MALDI-TOF MS could be adapted and used for the identification of molds in clinical routine diagnostics. Four reference strains (Aspergillus niger, A.fumigatus, A.terreus, A.flavus) and a clinical isolate (A.terreus) were examined. The preparation methods (I) complete formic acid extraction, (II) direct application and (III) suspension in distilled water were used for analysis of spores and frontmycelium from younger and older mold cultures. Two different masspektradatabases for species identification were compared; routine database BDAL and the specialized mold database, Filamentous Fungi Library. Also the collecting technique of mold prior to analysis with MALDI-TOF MS was evaluated. Sometimes, the species identification improved after extraction of mold cultures, while in other cases direct application was sufficient. Cultures with a lot of spores tended to give slightly more species identifications in BDAL regardless of the age of cultures. Filamentous Fungi Library, in some cases, tended to improve the performance compared to BDAL for younger cultures. More studies are required to evaluate and optimize MALDI-TOF MS as a method of mold identification.
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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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SQUEO, VALERIA. "Alterazioni del peptidoma serico di pazienti con tumore renale valutate tramite "label-free" (nLC-ESI-MS/MS) e "peptide profiling" (MALDI-MS/MS)." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/43783.
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Il progetto di questa ricerca è stato rivolto allo studio e alla caratterizzazione del peptidoma serico di soggetti sani e pazienti con carcinoma renale, così come alla diagnosi differenziale delle forme di tumore renale maligno da lesioni benigne. A questo proposito sono stati purificati, mediante tecnica ClinProt che utilizza microparticelle
magnetiche funzionalizzate, il siero di un ampio numero di pazienti affetti da ccRCC e soggetti controllo, estendendo lo studio anche a pazienti affetti da non-ccRCC. Le analisi di profiling peptidico sono state condotte mediante SM MALDI-TOF seguita da elaborazione dei profili spettrali per all’individuazione di clusters con potenziale diagnostico. Inoltre si è cercato di identificare proteine/peptidi con alterata espressione serica. A questo scopo sono state effettuate analisi mediante tecnologia nLC-ESI MS/MS e MALDI-TOF MS/MS. I risultati del "profiling" tramite MALDI-TOF sono stati verificati utilizzando un approccio proteomico diverso basato su una quantificazione relativa label-free in nLC-ESI MS/MS.
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Kempka, Martin. "Improved mass accuracy in MALDI-TOF-MS analysis." Licentiate thesis, Stockholm : Division of Analytical Chemistry, Royal Institute of Technology, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-313.
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Kriegsmann, Mark. "MALDI MS Imaging zur Untersuchung von synovialem Gewebe." Doctoral thesis, Universitätsbibliothek Leipzig, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-118897.
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Halgunset, Anders. "Typing av Legionella pneumophila med MALDI-TOF MS." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for bioteknologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-24697.
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Legionella pneumophila er fakultativ intracellulære bakterier som lever naturlig i akvatiske habitater. De kan replikere i makrofager og protozoer, bli overført til mennesker gjennom areosoler og føre til smitte. Ved eventuelle utbrudd er det viktig å kunne identifisere kilden, noe som i dag bli gjort ved å typebestemme L. pneumophila med sekvensbasert typing (SBT). Samtidig har matrix-assisted laser desorption/ionization ? time-of-flight (MALDI?TOF) mass spectrometry (MS) vist seg å kunne brukes til rask og effektiv identifisering av en rekke mikroorganismer på artsnivå, deriblant Legionella. Hos enkelte andre mikroorganismer har MALDI?TOF MS også vist å kunne skille mellom underarter. MALDI?TOF MS ble i denne oppgaven benyttet for å undersøke om det var mulig å skille L. pneumophila fra hverandre på stammenivå ved hjelp av MALDI Biotyper 3.0 programvare. Standardprotokollen anbefalt av Bruker Daltonics for proteinekstraksjon fra mikroorganismer ble tilpasset/optimalisert for bruk på L. pneumophila, slik at reproduserbare massespektre ble oppnådd. Evalueringen viste at proteinekstraksjon fra ca 2 µl biologisk materiale med 10 µl acetonnitrill og 10 µl maursyre, ga best scoreverdi opp mot Bruker Daltonics referansebibliotek. Den optimale temperaturen og varigheten for dyrkning ble vist å være henholdsvis 37 ˚C og 48 ± 2 timer. Det ble vist at lagring av skåler/kolonier med L. pneumophila ved 37 ˚C førte til forandringer i massespektrene, mens lagring ved 20 ˚C ikke medførte slike forandringer og at stabile massespektre ble oppnådd etter seks dagers lagring.Det ble bygget opp et referansebibliotek i MALDI Biotyper 3.0 bestående av referansespekter/referansetopplister (MSP) fra til sammen 48 Legionella spp. hvorav 41 var L. pneumophila -isolater. Med referansebiblioteket ble det vist at MSP (dannet ved standard innstillinger) for mange L. pneumophila var for like hverandre til å gi identifisering mot egne MSP. En klyngeanalyse ble brukt i sammenligning av L. pneumophila mellom SBT og MSP fra referansebiblioteket dannet med MALDI?TOF MS. Sammenligningen viste at diversiteten i proteinsammensetningen hos L. pneumophila, representert som MSP, ikke ga samme oppløselighet som gjennom sekvensvariasjonene fra genene i SBT -analysen.
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Trim, Paul James. "MALDI-MS imaging for direct drug distribution analysis." Thesis, Sheffield Hallam University, 2009. http://shura.shu.ac.uk/20455/.
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MALDI Imaging has gained huge interest in the past few years with an ever increasing population of specialists choosing to investigate samples using MALDI imaging, including growing interest and financial backing from pharma and contract research organisations. Presented within this thesis is the development and application of MALDI imaging techniques for a variety of analytical problems. The use of various software packages have been employed in the interpretation of the data acquired from MALDI experiments including, the use of statistical analysis for the identification of ion of interest from 6 distinct brain regions and also for the identification of ions of interest associated with small molecule tumour markers. The advantages of MALDI-IMS-MSI as a further separation stage within MALDI-MSI have been shown. Demonstrated is a method for MALDI-IMS-MS imaging of endogenous lipids in healthy tissue and tumours, also demonstrated is the application of MALDI-IMS-MS to xenobiotic distribution studies, it has been clearly shown that ion mobility separation within MALDI-MSI experiments can improve the analysis of xenobiotics by removing any interfering ions. With instrumentation development for MALDI a high repetition rate Nd:YVO4 laser has been assessed as a possible method for decreasing acquisition time.
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Sun, Zhen. "Studies of Atmospheric Pressure Visible-Wavelength MALDI-MS." University of Toledo / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1333747638.
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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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Zhen, Liu [Verfasser]. "Novel approaches for quantitative analysis of small biomolecules in MALDI-MS and SALDI-MS / Liu Zhen." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2020. http://d-nb.info/1219904481/34.
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Bertilsson, Sarah. "Glycanmapping of glycoproteins with UPLC-FLR-MALDI/TOF-MS." Thesis, Uppsala universitet, Analytisk kemi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-228040.
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Dempwolf, Wibke. "MALDI-TOF in der kontrollierten radikalischen Polymerisation und Präpolymeranalyse." Clausthal-Zellerfeld Papierflieger, 2007. http://d-nb.info/990376834/04.
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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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Schumacher, Joshua. "Carbon Nanotube Enhanced MALDI MS: Increasing Sensitivity Through Sample Concentration." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3595.
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Matrix-assisted laser desorption/ionization (MALDI) is a technique used in mass spectrometry for the ionization of biomolecules. A matrix solution is mixed with the analyte molecules to be investigated, and then spotted onto a specialized MALDI plate. The solvents evaporate leaving only the re-crystallized matrix with analyte dispersed throughout the crystals. Sample ionization is accomplished with a laser in the MALDI instrument. The spot diameter of the target is usually several orders of magnitude larger than the diameter of the laser, making it necessary to perform multiple laser investigations to accurately evaluate the analyte in the target spot.
Experiments were performed to utilize patterned areas of carbon nanotubes to provide sites for preferential crystallization of the liquid matrix/analyte solution, which led to lateral concentration for non-aqueous based matrices and produced a final dried matrix/analyte spot that was approximately the diameter of the laser spot at the point of investigation. This work shows the results of using aligned carbon nanotubes as the substrate for the matrix/analyte deposition and demonstrates an increase in signal to noise ratio and an improved detection capability of low analyte concentrations compared to the standard MALDI preparation technique.
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Worster, Belinda Mary. "MALDI-TOF-MS and neuropeptide signalling in the nervous system." Thesis, University of Sussex, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360682.
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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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000854199.pdf: 5245942 bytes, checksum: 78603c065ca6ba2cb04fe0531fde9643 (MD5)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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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 MilagreBanca: 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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Earnshaw, Caroline Jane. "Sample preparation methodologies for MALDI-MS imaging and related topics." Thesis, Sheffield Hallam University, 2009. http://shura.shu.ac.uk/19590/.
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The diverse applications of MALDI MSI are explored in this thesis with an emphasis on the sample preparation procedure and method development for small molecule analysis for a range of samples. The two main themes that have been focussed on are the pharmaceutical and metabolomic applications of this state of the art technique. MALDI MSI has been evaluated as a technique for the detection and imaging of antiasthmatic compounds in lung tissue. Four compounds were assessed initially with conventional MALDI MS experiments, followed by both direct and indirect tissue imaging experiments. Pharmaceutical tablet formulations have also been assessed using MALDI MSI to map the active component throughout the excipients contained within the tablet providing information that is critical to the manufacturing process such as the homogeneity of the active pharmaceutical ingredient (API) throughout the tablet. MALDI MSI has been applied to the relatively new addition to the 'omics sciences, metabolomics. A non-targeted metabolomics approach has been used to study both plant and animal tissue in an attempt to gain a greater understanding of the complex biological processes that occur within both types of tissue. Wheat grain was used as the model system to conduct the experiments and evaluate the application of both UV MALDI MS and IR LDI MS for plant metabolomics. These techniques provided complementary information to published literature, however the novel aspect of this study was the incorporation of imaging experiments for UV MALDI MS; this allowed the metabolites to be visualised in the wheat grain section. MALDI MSI was also used to explore the differences between mice with chronic relapsing experimental autoimmune encephalomyelitis; the animal model of multiple sclerosis alongside healthy controls. Spinal cord samples were analysed and the main difference was tentatively attributed to choline levels.
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Niare, Sirama. "Identification du repas sanguin des moustiques par MALDI-TOF MS." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0575/document.
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Le MALDI-TOF MS (Matrix Assisted, Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) est une technique protéomique qui est utilisée en routine pour l’identification des microorganismes dans les laboratoires de microbiologie. Ainsi, dans ce travail nous avons évalué le MALDI-TOF MS pour l’identification du repas sanguin des moustiques. Dans la première partie de notre travail, une revue bibliographique a été effectuée sur les différentes méthodes (sérologiques, biologie moléculaire) connues dans les études de préférence trophiques des arthropodes. La deuxième partie fut l’optimisation du MALDI-TOF MS pour l’identification de l’origine du repas sanguin des moustiques. Pour l’optimisation, Anopheles gambiae Giles et Aedes albopictus ont été artificiellement nourris sur le sang de plusieurs hôtes vertébrés en utilisant l’appareil Hemotek durant deux heures sous les conditions standard. Nos résultats ont montré que la comparaison des spectres provenant des moustiques nourris sur le même type de sang révèle une grande reproductibilité des profils protéiques. L’interrogation des MS spectres contre la base de données a révélé une identification correcte de l'origine du repas sanguin pour les spécimens collectés moins de 24 heures après la prise du repas sanguin. Pour les échantillons collectés sur le terrain, le MALDI-TOF MS a permis de détecter dans le repas de sang des moustiques une grande diversité d’hôtes domestiques. En conséquence la technique MALDI-TOF MS serait un outil efficace pour les études de surveillance épidémiologique des maladies vectorielles et l'identification de la préférence trophique de spécimens fraichement gorgésMALDI-TOF MS (Matrix Assisted, Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) is a proteomic technique that routinely used for microorganisms identification in clinical microbiology laboratory. Recently, the MALDI-TOF MS was successfully used as a innovative tool for arthropod identification. Thus, in this work we evaluated the MALDI-TOF MS to identify the blood meal sources from engorged mosquitoes. In the first part of our work, a bibliographical review was carried out on the different methods (serological, molecular biology) known in the trophic preference determination of hematophagous arthropods. The second part was optimization of the MALDI-TOF MS for identifying the origin of the blood meal of mosquitoes. For optimization, the Anopheles gambiae Giles and Aedes albopictus were artificially fed on several vertebrate hosts blood using the Hemotek device for two hours under standard conditions. Our results showed intra-species reproducibility and inter-species specificity of MS spectra from mosquitoes engorged on the same or different vertebrate hosts. The MS spectra querying against the database reveal a correct identification of the the blood meal origin from the specimens collected less than 24 hours post-feeding. For field samples, MALDI-TOF MS allowed to detect the mosquitoes blood meal fed on wide variety of domestic hosts. Consequently the MALDI-TOF MS technique would be an effective tool for epidemiological surveys of vector-borne diseases and the identification of the trophic preference of mosquito freshly engorged
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Hamburg, Daisy-Malloy. "Biochemical and MALDI-MS methods for characterization of ribosomal proteins." Cincinnati, Ohio : University of Cincinnati, 2008. http://rave.ohiolink.edu/etdc/view.cgi?acc_num=ucin1204305343.
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Thesis (Ph. D.)--University of Cincinnati, 2008.Advisor: Patrick Limbach. Title from electronic thesis title page (viewed Apr. 23, 2009). Keywords: MALDI-MS; ribosome; ribosomal proteins. Includes abstract. Includes bibliographical references.
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Mitrovic, Bojan. "DEVELOPMENT OF IONIC POLYMER NANOBRUSHES WITH APPLICATION IN MALDI-MS." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/838.
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The focus of this dissertation is in the development and application of responsive polymer nanobrushes. Mainly, preparation of the polymer nanobrushes will center on the development of so called mixed polymer brushes composed of the ionic and neutral monomers. Such mixed polymer brushes have a range of applications and uses, and their development will be evaluated in terms of the environmental stimuli response. Once an essential insight of the brush behavior is gained, application will be evaluated in terms of the protein fractionation prior to analysis by Matrix Assisted Laser Desorption Ionization Mass Spectroscopy - MALDI-MS.
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Ghebreamlak, Weyni. "Identification of Trypsin Digested Transferrin using HPLC and MALDI-MS." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-266157.
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In this project, separation of trypsin digested transferrin (Tf) has been studied, using a RP HPLC- UV system equipped with a C18 column. 0.1% TFA/MQ-water and 90% MeOH were used as mobile phase A and mobile phase B, respectively. For economic reasons, the protein cytochrome c (cyt-C) was used to optimize the digestion procedure and LC system, before analysis of Tf. Four digestion methods were applied for analyzing cyt-C and Tf. The first method was digestion with no denaturing, reducing or alkylating agent. The other digestion methods used urea or heating as a denaturing agent, and lastly dithiothreitol (DTT) and iodoacetamide (IAA) as reducing and alkylating agent, respectively. The results from HPLC-UV showed that a gradient elution with a high concentration of organic solvent is favorable for the separation of cyt-C peptides. MALDI-MS was used to identify peptides, and the outcomes showed that denaturation by heat before digestion gave the best results.I detta projekt har separation av trypsin-klyvt transferrin (Tf) studerats, med användning av ett RP HPLC-UV system, som bestod av en C18 kolonn. 0,1% TFA/MQ-vatten och 90% MeOH användes som mobilfas A respektive mobilfas B. Av ekonomiska skäl användes proteinet cytokrom c (cyt-C) före analys av Tf för att optimera klyvningsprocessen och LC systemet. Fyra klyvningsmetoder studerades för analysering av cyt-C och Tf. Den första metoden innehöll inget denaturerande, reducerande eller alkylerande medel. De andra klyvningsmetoderna innehöll urea eller värme som denaturerande medel, och slutligen ditiotreitol (DTT) och jodacetamid (IAA) som reducerande respektive alkylerande medel. Resultaten från HPLC-UV visade att en gradienteluering med en hög koncentration av den organiska lösningen är gynnsam för separationen av peptiderna från cyt-C. MALDI-MS användes för att identifiera peptiderna, och resultaten visade att denaturering med värme före klyvning gav bäst resultat.
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Müller, Lukáš. "Analýza proteomu piva pomocí hmotnostní spektrometrie." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2009. http://www.nusl.cz/ntk/nusl-216454.
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The aim of presented diploma thesis was to characterize recent knowledge in the field of beer proteomics. The main part of this work was focused on modern instrumental methods of protein analysis, especially on protein identification by mass spectrometry. In experimental part proteins from selected beer samples were isolated, purified and separated by 2-D electrophoresis. The identification was performed by MALDI MS/MS and LC-MS/MS. Identified proteins were divided into 6 groups - serpines and protein Z, trypsine/-amylase inhibitors, yeast proteins, LTP protein, hordeins and other proteins. Proteomic analysis provided identification of proteins important for final analytical and sensory characteristics of the beer - for final beer quality and taste
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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 MilagreBanca: 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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Bunch, Josephine. "Detection and imaging of pharmaceutical compounds in skin by MALDI-MS." Thesis, Sheffield Hallam University, 2005. http://shura.shu.ac.uk/19408/.
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Mass spectrometric techniques have been developed recently for the examination of biological tissue samples. Tandem mass spectrometry has been employed for the detection of pharmaceutical compounds and also mass spectrometric 'images' have been produced which show the spatial distribution of peptides, proteins and drugs in tissue. In this thesis, a programme of method development for the detection and imaging of topically applied pharmaceutical compounds in porcine epidermal tissue by MALDI-TOF-MS is presented. Direct analysis of fresh tissue sections was compared with the analysis of tissue imprints formed by blotting onto a variety of substrates. The samples were coated with matrix material by a prototype electrospray deposition device. Analyses were performed on a linear time-of-flight (LaserTOF 1500, SAI) mass spectrometer. Direct analysis of tissue and analysis of the C18 blots gave irreproducible data. Problems with matrix layer in-homogeneity were experienced with nitrocellulose and polyvinyl difluoride (PVDF) membranes. Reproducible data were obtained by analysis of tissue imprints created on carbon and cellulose membranes. All subsequent work was conducted using an Applied Biosystem Qstar pulsar i hybrid quadrupole time-of-flight mass spectrometer fitted with an orthogonal MALDI ion source and ion imaging software. The advantages of superior mass accuracy and resolution with such an instrument configuration were investigated. Electrospray and airspray methods were compared for analysis of tissue imprinted carbon and cellulose membranes. A novel method of pre-coating cellulose membranes in matrix by airspray prior to the blotting procedure was developed. The method was found to retain the expected distribution of the analyte. Ion images demonstrating the permeation of the applied compound into the skin were achieved by imaging a cross sectional imprint of treated tissue on a cellulose membrane precoated in matrix material. A calibration graph for the determination of ketoconazole was prepared using the sodium adduct of the matrix ion as an internal standard. This enabled construction of a quantitative profile of drug in skin. Conventional haematoxylin and eosin staining and microscopy methods were employed to obtain a histological image of the porcine epidermal tissue. Super imposing the mass spectrometric and histological images revealed drug permeation into the dermal tissue layer. A quantitative corneum tape stripping/HPLC method was developed for comparison. Useful data was acquired and further work suggested to facilitate a full validation of the methods presented in this thesis.
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AL-Jabiry, Ekram. "Syntetisering av en ny MALDI-MS matris med användning av Suzukikopplingsreaktion." Thesis, Uppsala universitet, Institutionen för läkemedelskemi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-444539.
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Chen, Shuo. "MALDI-TOF MS data processing using wavelets, splines and clustering techniques." [Johnson City, Tenn. : East Tennessee State University], 2004. http://etd-submit.etsu.edu/etd/theses/available/etd-1112104-113123/unrestricted/ChenS121404f.pdf.
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Thesis (M.S.)--East Tennessee State University, 2004.Title from electronic submission form. ETSU ETD database URN: etd-1112104-113123 Includes bibliographical references. Also available via Internet at the UMI web site.
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Hamdi, Cassandra. "Clostridium difficile : Rapid typing Clostridium difficile using MALDI-TOF MS analysis." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17659.
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Ballade, Tosco Armelle. "Identification et caractérisation par spectrométrie de masse de substances à activités biologiques produites par des souches de BacillusS." Thesis, Bordeaux 1, 2011. http://www.theses.fr/2011BOR14240.
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Les substances synthétisées par les bactéries du genre Bacillus présentent un grand intérêt en raison de leurs nombreuses activités antimicrobiennes. L’objectif de ce travail a consisté à mettre au point un protocole analytique fiable pour extraire ces substances, et les caractériser par spectrométrie de masse et par leurs activités biologiques. Une première approche par spectrométrie de masse MALDI-ToF ne nous a pas donné de résultats satisfaisants en raison d’un phénomène de suppression spectrale. Une seconde stratégie d’étude combinant deux techniques analytiques a alors été envisagée. Les composés et les familles lipopeptidiques ont d’abord été séparés par chromatographie liquide analytique et analysés en ligne en mode de couplage ESI-IT. Ensuite les fractions collectées ont été caractérisées par spectrométrie de masse MALDI-Q-ToF (et ESI-Q-ToF) pour déterminer les masses exactes et obtenir des spectres de fragmentation complémentaires des premiers. Ce procédé nous a permis d’établir des critères d’identification des composés et des familles de lipopeptides de structures connues. Cette caractérisation est basée sur des temps de rétention en chromatographie, des mesures de masses exactes et sur des schémas de fragmentations. Nous avons fait de même avec les composés non identifiés et ensuite évalué leur activité biologique en réalisant des tests de diffusion sur agar. Cette stratégie d’étude permet de faire un criblage de l’ensemble des biomolécules produites par des souches de Bacillus et de relier une structure à une activité biologique. Ce protocole, mis au point pour des cultures réalisées en milieu liquide, a ensuite été adapté aux cultures sur boîtes de Pétri pour pouvoir analyser les composés produits à l’échelle de la colonie bactérienneCompounds produced by Bacillus bacteria present a major interest because of their biological activities. The aim of this work was to develop a reliable analytical methodology to extract these compounds, and to characterize them by mass spectrometry and by their biological activities. A first approach by MALDI-ToF mass spectrometry doesn’t give satisfactory results because of strong spectral suppression effects. Therefore, we have designed a second strategy which combined two analytical technologies. First, compounds and lipopeptides families were separated by analytical liquid chromatography and analysed by ESI-IT. Then, collected fractions were characterized by MALDI-Q-ToF (and ESI-Q-ToF) in order to determine accurate mass measurements and obtain complementary product ion spectra. This process led us to establish identification criteria of compounds and lipopeptides families which have known structures. This characterization is based of retention times in chromatography, accrurate mass measurements and fragmentation schemes. We have achieved the same experiments with non identified compounds and then assessed their biological activity by agar well diffusion test.This strategy allows to obtain a screening of whole of the biomolecules produced by bacillus strains and establishes a link between structure and biological activity. This methodology, designed for cultures in liquid medium, was then adapted to cultures in Petri disches in order to analyse compounds producted at the bacterial colony scale
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PENG, LIJUAN. "MATRIX-ASSISTED LASER DESORPTION/IONIZATION (MALDI) TARGET MODIFICATION FOR ENHANCED PROTEOMICS ANALYSIS AND PLASMA POLYMER CHARACTERIZATION BY MALDI MASS SPECTROMETRY." OpenSIUC, 2010. https://opensiuc.lib.siu.edu/dissertations/207.
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The work described in this dissertation is divided into three sections. In the first section three surface modifications are used to produce MALDI targets having reduced surface-protein binding affinity with a goal of increasing peptide/protein MALDI ion signals and lowering the limits of detection (LODs) for proteins and peptides. The second section discusses a bioselective MALDI target, produced via radio frequency (rf) plasma deposited ethylenediamine (EDA), for on-target separation of complex protein mixtures. The third section develops a new approach for characterization of rf plasma-deposited bulk polymers by using MALDI MS. Previous studies in our group have shown that the analyte signal in a MALDI MS experiment is strongly influenced by the binding interactions between the target surface and the analyte. Specifically, the analyte signal increases with decreasing surface-analyte binding affinity, which has been attributed to more unbound analyte being available for incorporation within the MALDI matrix. In the presented studies MALDI targets are modified with polyethylene glycol (PEG)-like structures via chemical grafting of PEG onto polyurethane (PU) film and rf plasma polymerization of ethylene oxide vinyl ether (EO2) and tetraglyme. It is shown that there are enhancements in the protein MALDI ion signals on these modified targets and that the LOD for target proteins is decreased by a factor of 2-10 in comparison with the conventional stainless steel MALDI target. On-probe affinity capture (OPAC) MALDI MS, developed in our group, has shown that functional group modified MALDI targets can be used to rapidly and selectively isolate target analytes from complex samples. For applications involving analysis of complex peptide/protein mixtures, fractionation of the mixture on the basis of component pI can reduce MALDI ion suppression effects leading to efficient ionization of larger numbers of mixture components. In the present studies a MALDI target is modified by rf plasma deposition of polymerized EDA to yield an OPAC target suitable for capture of proteins with low pI (expected to be negatively charged at neutral pH). In subsequent MALDI MS analyses of both control and biological mixtures after fractionation on the OPAC target it is observed that a significant number of additional peptide/protein ion signals are detected. The results of these studies, along with studies of the effects of the density of the primary amine functionality on the bio-selective MALDI ion signals, are presented. The complex nature of the polymer films resulting from plasma polymerization makes it very difficult to characterize their molecular structures. The presented study is the first to use MALDI MS for characterization of rf plasma-deposited bulk polymers and for investigation of the rf plasma polymerization process. It is shown that the mass spectra of the soluble fraction of allyl alcohol, EO2 and ethylene glycol butyl vinyl ether -plasma polymers contain clear polymer series. Furthermore, it is found that the peaks of the EO2-plasma polymer series shift to higher molecular weight distribution with decreasing plasma duty cycle. In contrast to predictions based on conventional radical polymerization, the mass spectra of all three plasma polymers exhibit the same repeat unit of 44 Da, for which the most likely structure would be -(CH2CH2O)-.
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Shenar, Nawar. "Spectrométrie de masse par désorption/ionisation laser de peptides modèles : applications en protéomique." Montpellier 2, 2008. http://www.theses.fr/2008MON20150.
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Jemmali, Zaïneb. "Développements méthodologiques en TLC/MALDITOF MS et GC/MS pour l’analyse des composés terpénoïdes présents dans les résines végétales." Thesis, Orléans, 2016. http://www.theses.fr/2016ORLE2061/document.
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Les résines végétales sont des sécrétions de végétaux qui ont été utilisées par l’homme de l’Antiquité à nos jours dans de nombreuses applications (pharmaceutique, cosmétique et artistique). Ces exsudats sont composés majoritairement de terpènes. L'identification et la quantification de l'ensemble de ces composés dans les extraits végétaux reste un défi du fait de leur très grande diversité structurale. L’objectif de ce travail a été de développer de nouvelles approches analytiques pour identifier et quantifier les composés terpéniques présents dans ce matériel végétal afin d’en assurer le contrôle qualité et la certification. Deux méthodes séparatives ont été sélectionnées: la TLC et la GC. Pour ces deux techniques on s’est intéressé à toutes les potentialités de leur couplage avec la spectrométrie de masse. Le développement en TLC-1D et TLC-2D a permis le « screening » rapide des résines végétales et la faisabilité du couplage avec le MALDI-TOF-MS a été mise en évidence pour l’identification des marqueurs majoritaires (acides triterpéniques). La GC a permis une caractérisation plus aboutie des résines en mettant en place une méthode d’analyse exhaustive des terpènes des plus volatils au non-volatils. L’optimisation des différentes étapes de la méthodologie GC-MS s’est effectuée en se basant sur la méthode des plans d’expérience ainsi que sur des analyses statistiques tels que l’ACP et la CAH. Dans un souci d’apporter des éléments plus précis pour distinguer les résines les plus proches, la quantification de leurs marqueurs majoritaires a été établie après une validation complète de la méthode GC. L’ensemble de ce travail a permis de développer des outils pour une caractérisation rapide des extraits de résines permettant de différencier les espèces même les plus prochesResins are hydrocarbon secretions of many plants and well known for their protective benefits. They have been used as raw materials for a wide range of applications (pharmaceutic, cosmetic and artistic). Plant resins are complex mixtures of organic substances mainly terpenoid compounds which constitute the most abundant and structurally diverse group of plant secondary metabolites. The chemical characterization of this material results in long and difficult separation due to the wide range of polarity and volatility of its constituents. The aim of this work was to develop new analytical approaches to improve the identification of resins certifying their origin and ensuring the quality control. For that purpose two analytical methods were selected: TLC and GC approaches hyphenated to mass spectrometry. TLC-1D and TLC-2D allow a rapid screening and first visual differences of resins. The innovating TLC coupling to MALDI-TOF-MS gives a clear identification of major markers (triterpenic acids). In order to have complementary information about the composition of resins, a gas chromatography-mass spectrometry (GC-MS) method was developed to analyze volatile to non-volatile compounds. The various stages of optimization were based on experimental design and statistical (PCA and HAC) approaches. For closely related resins, a quantitative approach was investigated based on a complete validation for major markers. This work allows the development of two complementary techniques that give a powerful approach for fast and reliable differentiation of various resins even the closest ones
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Elvingson, Ebba. "Art- och genusbestämning av bakterier direkt från blododlingar med MALDI-TOF MS." Thesis, Linnéuniversitetet, Institutionen för kemi och biomedicin (KOB), 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-36355.
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Sepsis är ett allvarligt tillstånd som uppstår när bakterier går från vävnad till blodbanan. Positiva blododlingar odlas på agarplattor och bakterier analyseras med Matrix Assisted Laser Desorption Ionisation Time-of-flight Mass spectrometry (MALDI-TOF MS) där prov blandas med en matrix och sedan bestrålas med laser. Proteinerna i provet joniseras och rör sig mot en detektor, vilket ger ett m/z-spektrum som jämförs med referensspektrum i en databas och ett score-värde erhålls över hur väl analyten liknar referensen. Arbetets syfte var att undersöka möjligheten att direktidentifiera bakterier från blod med en viss preparation innan analys med MALDI-TOF MS och på så vis möjliggöra snabbare preliminära svar samt undersöka möjligheten att särskilja Staphylocoocus aureus och koagulasnegativa stafylokocker. Innan analys med MALDI-TOF MS centrifugerades blod från positiva blododlingar blod i flera steg med 5 % natriumkloridlösning (NaCl-metoden). Dessutom testades ett kommersiellt kit (Sepsityper, Bruker Daltonics). Med NaCl-metoden sågs korrekt identifiering hos 66 % av inokulerade proverna. Av blododlingar innehållande med S. aureus respektive koagulasnegativa stafylokocker identifierades 60 % respektive 43 % av bakterierna korrekt. Med Sepsiptyper erhöll 58 % av proverna godkänt score-värde. Slutsatsen blev att det är möjligt att identifiera bakterier direkt från blod efter viss preparation, men metoden bör utvecklas mer då det fanns en signifikant skillnad i score mellan NaCl-metoden och nuvarande metod. Det är dock möjligt att skilja mellan Staphylococcus aureus och koagulasnegativa stafylokocker. Fler studier är nödvändiga för att avgöra möjligheten att föra in någon av metoderna i rutindiagnostiken.
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Liti, Samone. "Evaluation of sample preparation techniques for MALDI-TOF-MS analysis of oligosaccharides." Thesis, Uppsala universitet, Avdelningen för analytisk farmaceutisk kemi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-277957.
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The aim of this study was to optimize the sample preparation and methods for analysis of oligosaccharides of hyaluronic acid with MALDI- TOF-MS. The analysis was carried out on an Autoflex speed MADLI-TOF- MS instrument with both linear and reflectron mode. Matrices used in this study were 2,5-DHB and Super-DHB and type of matrix was chosen depending on the size of the analyzed oligosaccharides. The application of sample and matrix on the target that gave the most homogenous crystallization was sandwich and the laser power in the MALDI was kept at 65 %. Since it is known that salts and buffers interfere with the analysis, sample clean-up such as solid phase extraction (SPE) in pipette tips and dialysis was performed. SPE worked best for low mass oligosaccharides and provided high intensity and little noise. With SPE a concentration of the analyte could be done which was the advantage over dialysis. Dialysis worked well for larger oligosaccharides and mixtures of different sized oligosaccharides. Another way of using MALDI for biomolecule analysis is with TLC-MALDI. A fast and accurate separation was achieved and analysis could be done directly from the plate. The optimized methods were evaluated according to linearity and precision, LOD, mass accuracy and matrix stability. The linearity and precision was good in a higher concentration range (50 μg/mL and higher), but the test for limit-of-detection (LOD) indicated that concentrations from 20-30 μg/mL could be analyzed with no interference from the background. The mass accuracy was within the acceptable limits according to Bruker Daltonics when a mass calibration was done for each analyzed sample. The stability of the matrix in solution was difficult to study because of the day-to-day variation in intensities given by the MALDI-TOF-MS technique.
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Hart, Philippa Jayne. "MALDI-MS investigation of skin and its response to irritants and sensitisers." Thesis, Sheffield Hallam University, 2012. http://shura.shu.ac.uk/20695/.
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There is increasing interest in in-vitro methodology for testing chemical toxicity, one of the drivers for this is European legislation; specifically Directive 76/768 EEC, which prohibits animal testing in the cosmetic industry. In skin toxicological testing, an alternative methodology to in-vivo irritancy experiments already available uses a synthetic skin model and involves measurement of cell viability and release of cytokine interleukin (IL)-1a. There is currently no fully validated in-vitro test for sensitization, although there are a number that are in development. Some of these include the human cell line activation test (h-CLAT) (Sakaguchi et al., 2010), the direct peptide reactivity assay (DPRA) (Gerberick et al., 2009) and the myeloid U937 skin sensitization test (MUSST) (Ade et al., 2006). This study utilises ex-vivo human skin as an initial platform for method development. Areas of analysis have included the lipidomic and proteomic responses of ex-vivo human skin to sensitizers and irritants. Matrix assisted laser desorption ionisation-ion mobility separation-mass spectrometry (MALDI-IMS-MS) was used to identify peptides and lipids in-situ. Ion mobility separation allowed for discrimination between isobaric species due to the selection of specific precursor ions and cleaner MS/MS spectra. Many of the peptides identified belonged to serum albumin, keratin and collagen protein families, which are known to be abundant within human skin. Lipids identified included: sphingomyelin, glycerophospholipids, ceramide species anddi/triglycerides. In skin studies performed in other research groups, sphingomyelin and ceramide species were found to change expression in response to initiation of inflammation, thus making them of biological significance when investigating lipidomic responses to chemical sensitizers and irritants. Tryptic peptide MALDI-MS images have shown differences in human skin, as a result of chemical exposure. MALDI-MS images at 150pm and 30pm resolution have provided information on the localisation of detected species. Changes in expression of species in treated and untreated samples, as well as between different layers of human skin have also been visualised via multivariate statistical analyses. These analyses of ex-vivo human skin have demonstrated that MALDI-IMS-MS is a technique which can be used to detect changes in protein/peptide and lipid profiles in response to sensitisers and irritants. The technique also allows for the localisation of species detected within the different layers of human skin.
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Ye, LiYun. "Characterization of A-type Proanthocyanidins in Peanut Skins Using MALDI-TOF MS." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/72283.
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Peanut skin, a low-value agriculture waste product, has drawn lots of research interest in recent years, due to its high content of A-type proanthocyanidins. A-type proanthocyanidins have been believed to contribute to cranberries' anti-UTI (urinary tract infection) effect. In this study, we compared the A-type proanthocyanidins in cranberry and peanut skin crude extracts using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Many similarities were found in the proanthocyanidin composition of cranberries and peanut skins. New oligomeric and polymeric proanthocyanidins in peanut skins, including heteroproanthocyanidins and proanthocyanidins with sugar moieties or galloyl esters, were tentatively identified. Solid phase extraction (SPE) and HPLC fractionation largely improved MALDI-TOF's ability to detect proanthocyanidins with high degrees of polymerization (DP). By analyzing the identified compounds in each fraction, we were also able to find some interesting elution pattern of the proanthocyanidins on the SPE cartridges and on the HPLC column. For example, the elution order on both the SPE cartridges and the diol phase column generally followed the DP. A-type proanthocyanidins tended to elute earlier than the B-type. Prodelphinidins retained much longer than other proanthocyanidins with the same DP. These findings may help researcher to identify future research directions and develop new separation methods to facilitate the identification of bioactive components in proanthocyanidin-rich plant extracts.Ph. D.
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Tummala, Manorama. "Surfactant-Aided Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (SA-MALDI MS)." University of Cincinnati / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1100672049.
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Nakano, Satoshi. "Development and evaluation of MALDI-TOF MS-based serotyping for Streptococcus pneumoniae." Kyoto University, 2016. http://hdl.handle.net/2433/215402.
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Norrman, Cassandra. "Validering av online databas för identifiering av svamp med MALDI-TOF MS." Thesis, Umeå universitet, Biomedicinsk laboratorievetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-160083.
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Marques, Lygia de Azevedo. "Aplicação de tecnicas avançadas de espectrometria de massas em ciencias de alimentos e perfumaria." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/248689.
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Orientador: Marcos Nogueira EberlinDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica
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Resumo: Neste trabalho aplicamos técnicas avançadas de espectrometria de massas, (MALDI-TOF e ESI-MS) na análise de micotoxinas em alimentos e na tipificação e verificação de fraudes em perfumes. Aplicamos a técnica MALDI-TOF em análises de micotoxinas, e esta mostrou excelente desempenho nas análises de aflatoxinas e ocratoxina e vantagem sobre a técnica de escolha atual, o método ELISA. Esta vantagem é principalmente maior especificidade através de maior exatidão em medidas de massas e, portanto, maior confiabilidade. O Planejamento de experimento foi uma ferramenta valiosa para obtenção das melhores condições e estudo dos parâmetros de interferência. O limite de detecção encontrado para a técnica foi da ordem de 25 pg para aflatoxinas e de 1 ng para ocratoxina, com perspectiva de melhoria através de aumento da massa amostral em estudos futuros para adaptação da metodologia de extração na matriz de interesse à técnica MALDI-TOF. A técnica ESI-MS foi utilizada para a tipificação e detecção de perfumes proporcionando, através da análise de componentes principais (PCA), a diferenciação com segurança entre perfumes originais, falsos e inspirados, utilizando como indicadores componentes polares não majoritários característicos de cada categoria avaliada. Este estudo abre caminho para que esta técnica seja utilizada na avaliação de perfumes que estão sob suspeita de falsificação com auxilio de uma biblioteca de "fingerprint" de perfumes por ESI-MS. O emprego da técnica de MALDI-TOF também é uma opção vantajosa para o monitoramento da qualidade de grãos quanto a presença de toxinas indesejáveis, bem como ameaças de bioterrorismo.
Abstract: In this work we applied advanced mass spectrometry techniques (MALDI-TOF and ESI-MS) to micotoxin analysis in food and for the typification and detection of counterfeit perfumes. MALDI-TOF was applied to micotoxin analysis, which showed excellent performance for the analysis of aflatoxins and ochratoxin with advantage over the current technique of choice, the ELISA method. This advantage is mainly its greater specifity due the exactness of the measurements, therefore with higher reliability. The surface analysis was a valuable tool to attain the best conditions and study the interference of several parameters. The detection limit found for the technique was 25 pg for aflatoxins and 1 ng for ochratoxins, with perspective of improvement through increase of the sample mass in future studies for adaptation of the methodology of extration in the matrix of interest for the MALDI-TOF technique. The ESI-MS technique was used for typification and detection of counterfeit perfumes, providing, through principal component analysis (PCA), the characterization of original, counterfeit and inspired perfumes, using as minoritarian polar compounds as diagnostic ions of each perfume category evaluated. We envisage that the method can be used to establish a ESI-MS fingerprinting library of perfumes for comparison with those from samples under investigation, and that such a library could be updated constantly by the addition of ESI-MS of new perfumes even before they are commercially released. MALDI-TOF technique is also an advantageous option for the monitoring of crop quality relating to the presence of undesirable toxins, as well as bioterrorism threats by micotoxin poisoning.
Mestrado
Quimica Analitica
Mestre em Química
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Rivas, Becerra Daniel. "Aplicación de MALDI-TOF Imaging y HPLC-MS/MS al estudio de la degradación del polímero policaprolactonadiol en diferentes medios acuáticos." Doctoral thesis, Universitat Politècnica de Catalunya, 2017. http://hdl.handle.net/10803/461713.
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Water is essential for life, human activities and ecosystems. The global climate change together the population increase causes an imbalance between the demand of water and the available water resources.. All these factors lead to a increasing deterioration of human health and aquatic ecosystems.
The characterization of aquatic systems includes structural and functional aspects. In addition, it is necessary to emphasize the degradation of natural and anthropogenic organic matter Aquatic ecosystem ecology, to study the loss of organic matter, uses the weight loss of natural organic devices such as dry leaves or fragments of standardized wood that has been exposed to the environment.
Bearing this in mind, the main objective of this thesis was to add more chemistry knowledge into the river ecology by using of a synthetic material probe to study its degradation in natural and engineering aquatic environments. The device is based on a commercial polymer exposed for a while to different aquatic environments. The degradation of the synthetic material probe is collected and analyzed by using advanced mass spectrometry techniques such HPLC-MS, MALDI-TOF/MS and MALDI IMAGING.
Chapter 1 (Introduction) is distributed into three parts. The first presents the problems associated with the quality of the aquatic environment, their effects on ecosystems and succinctly describes the biotic and abiotic processes of degradation of organic matter and its application in WWTPs. In the second part there is a brief description of the polymers, their uses and degradation, as well as the analytical techniques used for their characterization. In the third part, we describe the mass spectrometry analytical techniques used in this thesis, emphasizing those that allow the study of space in two dimensions, through the corresponding generation of images (IMAGING).
The main objectives of the thesis are described in Chapter 2.
Chapter 3 describes the selection of possible polymers to be used, and the optimization of the analytical methodologies used by MALDI-TOF/MS. The chosen polymer was (polycaprolactone 1250). Probes consisting of a plastic capsule-piston with a sample of the polymer were prepared. Exposure experiments were carried out at various points of the Ebro River. The results obtained showed different types of degradation. Finally, these results were compared with those obtained in parallel using classical methods (using tree leaves). Chapter 4 describes laboratory-scale experiments performed with the selected polymer. These experiments consisted in exposing polymer samples for a time in aqueous systems, in sterile, aerobic and denitrifying conditions. The use of the novel MALDI IMAGING technique allowed the observation of degradation differences along the surface of the sample between the three types of conditions tested, obtaining images of the most interesting ions. Likewise, the statistical treatment of the results obtained was confirmed by the results of the images.
The success of the previous laboratory-scale experiment drove it to the next level and this experiment was applied it to wastewater samples. The WWTP El Prat de Llobregat was the site chosen, placing polymer probes in the secondary reactor, where the aerobic and denitrification (anaerobic) treatment are performed. The MALDI IMAGING technique was used to analyze the samples and high-resolution liquid chromatography coupled to mass spectrometry (HPLC-MS) was used as a complementary technique to elucidate the structures of the degradation compounds. The results obtained allowed to establish the degradation mechanisms and corresponding transformation products differentiated between the two types of environments studied. The description and results of this study is presented in Chapter 5.
Finally, in chapter 6 contains a general discussion of each experiment of this thesis.El agua es esencial para la vida y las actividades del ser humano y de los ecosistemas. El cambio global climático junto al incremento constante de población hace que el desequilibrio entre la demanda de agua y los recursos hídricos disponibles se incremente. Todo ello redunda en un impacto creciente sobre la salud humana y los ecosistemas acuáticos. La caracterización de los sistemas acuáticos incluye tanto aspectos estructurales como funcionales. Entre estos últimos hay que destacar la degradación de la materia orgánica, tanto natural como de origen antropogénico. La ecología de sistemas acuáticos utiliza la pérdida de peso de soportes orgánicos naturales como hojas secas o fragmentos de madera estandarizados expuestos al medio para calcular la pérdida de materia orgánica. Partiendo de esta idea el objetivo principal de esta tesis ha sido introducir más conocimiento químico dentro de la ecología de ríos utilizando una sonda basada en material sintético para estudiar su degradación en ambientes acuáticos, tanto naturales como ingenieriles. El dispositivo contiene un polímero comercial, el cual después de estar expuesto un tiempo en diferentes entornos acuáticos se recoge y se analiza su degradación utilizando técnicas avanzadas de espectrometría de masas, como son HPLC-MS, MALDI-TOF/MS y MALDI IMAGING. El capítulo 1 (Introducción) está dividido en tres partes. En la primera se presenta la problemática asociada a la calidad del medio acuático, sus efectos sobre los ecosistemas y se describen sucintamente los procesos bióticos y abióticos de degradación de la materia orgánica y su aplicación en las EDAR. En la segunda parte se realiza una breve descripción de los polímeros, sus usos y degradación, así como las técnicas analíticas que se usan para caracterizarlos. En la tercera parte, se describen las técnicas analíticas de espectrometría de masas empleadas en esta tesis, haciendo énfasis en aquellas que permiten el estudio espacial en dos dimensiones, mediante la correspondiente generación de imágenes (IMAGING). Los objetivos principales de la tesis se detallan en el capítulo 2. En el capítulo 3 se describe la selección de posibles polímeros a utilizar, así como la optimización de los correspondientes métodos de análisis mediante MALDI-TOF/MS. Con el polímero finalmente elegido (policaprolactonadiol 1250) se prepararon sondas poliméricas y se realizaron experimentos de exposición en diversos puntos del río Ebro. Los resultados obtenidos evidenciaron diferentes tipos de degradación. Finalmente, éstos se compararon con los obtenidos en paralelo mediante métodos clásicos, empleando hojas de árbol. Con el polímero seleccionado se realizaron experimentos a escala de laboratorio, que se describen en el capítulo 4, consistentes en la exposición de muestras de polímero durante un tiempo concreto en sistemas acuosos en condiciones estériles, aeróbicas y desnitrificantes. El uso de la novedosa técnica MALDI IMAGING, permitió observar diferencias de degradación a lo largo de la superficie de la muestra entre los tres tipos de condiciones ensayadas, obteniendo imágenes de los iones más interesantes. Así mismo, el tratamiento estadístico de los resultados obtenidos confirmó las imágenes adquiridas. El éxito del experimento anterior a escala de laboratorio, impulsó llevarlo al siguiente nivel y aplicarlo en muestras de aguas residuales. Así pues, las sondas de polímero se instalaron en el reactor secundario de la EDAR del Prat de Llobregat, donde se realiza un tratamiento de nitrificación (aerobio) y uno de desnitrificación (anaerobio). Para analizar las muestras se utilizó la técnica MALDI IMAGING y HPLC-MS para elucidar las estructuras de los compuestos de degradación y establecer los mecanismos de degradación correspondientes productos de transformación en función de las condiciones estudiadas. Finalmente en el capítulo 6 se comenta la discusión general de cada experimento realizado extrayéndose unas conclusiones finales.
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Matajira, Carlos Emilio Cabrera. "Identificação de estirpes do gênero Streptococcus pela técnica de reação em cadeia da polimerase (PCR) e espectrometria de massa MALDI-TOF." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-27102015-082622/.
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Métodos microbiológicos tradicionais como isolamento, coloração de Gram e testes bioquímicos auxiliam na identificação do gênero Streptococcus, no entanto, as espécies apresentam ampla variação fenotípica, tornando difícil a identificação ou diferenciação das mesmas apenas por estes métodos. Uma das espécies mais importantes em suínos, Streptococcus suis, tem provocado grandes prejuízos em todo o mundo e tem sido descrito como uma importante zoonose em alguns países. S. suis está presente nas vias respiratórias superiores, colonizando principalmente tonsilas, cavidades oral e nasal facilitando a alta disseminação por contato direto, principalmente em leitões entre 4 e 12 semanas de vida. Os quadros clínicos mais frequentes em suínos infectados pelo S. suis são meningite, artrite e pneumonia. O objetivo do presente estudo foi identificar estirpes do gênero Streptococcus mediante as técnicas de reação em cadeia pela polimerase (PCR), sequenciamento parcial do gene 16S rRNA e espectrometria de massa MALDI-TOF (MALDI-TOF MS). As análises por PCR e por MALDI-TOF MS resultaram na identificação de 215 estirpes como S. suis e 35 como diferentes espécies pertencentes ao gênero Streptococcus. Os resultados da identificação das 35 estirpes pertencentes a outras espécies do gênero Streptococcus pelo MALDI-TOF MS foram confirmados pelo sequenciamento parcial do gene 16S rRNA, sendo que as duas técnicas apresentaram 100% de concordância. Os resultados obtidos indicam grande eficácia na utilização das técnicas avaliadas para a identificação de S suis e de outras espécies do gênero Streptococcus. A técnica de MALDI-TOF MS, apesar do custo elevado do equipamento, apresentou a vantagem de ser rápida, apresentar baixo custo por análise e reduzida utilização de materialTraditional microbiological methods such as isolation, Gram staining and biochemical tests help to identify the Streptococcus genus, however, the species present broad phenotypic variation, making it difficult for their identification or even differentiation just by these methods. One of the most important species in swine, Streptococcus suis, has led to great losses worldwide and has been described as an important zoonosis in some countries. S. suis is present in the upper airways, especially colonizing tonsils, oral and nasal cavities facilitating the high dissemination by direct contact, especially among piglets between 4 to 12 weeks of age. The most common clinical manifestations in pigs infected by S. suis are meningitis, arthritis and pneumonia. The aim of this study was to identify Streptococcus strains by polymerase chain reaction (PCR), 16S rRNA gene partial sequencing and MALDI-TOF mass spectrometry (MALDI-TOF MS). PCR and MALDI-TOF MS analysis resulted in the identification of 215 strains as S. suis and 35 as different species of the Streptococcus genus. The identification of the 35 strains belonging to other species of the genus by MALDI-TOF MS was confirmed by 16S rRNA gene partial sequencing, and both techniques presented 100% concordance. These results demonstrate the high efficiency in the use of the evaluated techniques for the identification of S. suis and the other species of the Streptococcus genus. The MALDI-TOF MS technique, despite the equipment high cost, presented the advantage of being fast, have low cost per analysis and reduced material usage
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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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Lyrio, Tenorio Correia Carolina. "Le marquage des peptides avec des métaux et détection par MS et l'optimisation des procédures de l'extraction de métalloprotéin dans les échantillons biologiques à des fins de protéomique." Thesis, Pau, 2014. http://www.theses.fr/2014PAUU3008/document.
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Ce travail a développé une nouvelle méthode pour l'identification et la quantification des peptides, par l'optimisation de certaines stratégies disponibles appropriées pour le marquage des peptides avec des métaux lanthanide, une séparation par nano-HPLC et détection UV, et suivi par MALDI MS. Tout d'abord, les peptides ont été marqués avec les trois métaux lanthanides différents et un réactif fonctionnel - DOTA. Les résultats montrent que la réaction de transformation en dérivé à l'aide du réactif chélateur DOTA-NHS-ester a été efficace pour des peptides individuels et des mélanges de peptides, vérifiées à partir de la relation m/z obtenue par MALDI MS. L'application optimisée d’un complexe (Cytochrome C digest) a montré des résultats comparables à ceux obtenus avec des peptides modèles. En parallèle, nous avons effectué l’optimisation pour la purification de métalloprotéine dans la bile de poisson, qui est signalée entant que biomarqueurs de contamination métallique de l'environnement. Des procédures différentes (différents moments de centrifugation et différentes températures de traitement thermique) et les agents (DTT, β-mercaptoéthanol et TCEP) réduisant ont été apliqués pour purifier les MT isolées de la bile et du foie des poissons (Oreochromis niloticus). Des analyses spectrophotométriques ont été utilisées pour quantifier les échantillons de MT, et le gel SDS-PAGE a été utilisé pour évaluer qualitativement les différents résultats de la procédure. Chaque procédure a en suíte été évaluée statistiquement, une méhtode des surfaces de réponse a été appliquée. Les MT de la bile semblent être plus adéquate pour la surveillance de l'environnement en ce qui concerne l'exposition récente à des xénobiotiques qui peuvent influer sur l'expression protéomique et metalloproteomique de cette matrice biologique. Une procédure d’exposition à des métaux dans le laboratoire a montré que les métaux étaient significativement importante pour l’évaluation de la contamination à partir de la quantification de MT, selon le traitement de données par une techinique de réseau neuralThis work developed a new method for the identification and quantification of peptides, by optimizing some of the available strategies suitable for labeling peptides with lanthanide metals with subsequent separation by nano-HPLC with UV detection, matrix-assisted laser desorption ionization-mass spectrometry (MALDI MS). First, peptides were labeled with the three different lanthanide metals using a functional DOTA-based reagent. The results demonstrate that the derivatization reaction using the chelating reagent DOTA-NHS-ester was effective for single peptides and peptide mixtures, verified from the m/z relation obtained by MALDI MS. The application of the optimized method in a more complex matrix (Cytochrome C digest) showed results comparable to those obtained with model peptides. In parallel, environmental analyses were conducted, by performing the standardization of metalloprotein purification in fish bile, since this matrix has been reported as a biomarker for environmental metal contamination. Different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, β-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated. A response surface methodology was applied for bile samples, in order to further evaluate the responses for this matrix. In an environmental context, biliary MT was lower than liver MT, and, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix. A procedure for exposure to metals in the laboratory showed that some metals are significantly important for the assessment of contamination from the quantification of MT, according to the data processing by atifical neural network (ANN)
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Motari, Edwin Mwamba. "Structural Studies of Oligosaccharides Attached to Proteins Expressed in Different Organisms and PEGylation of a non-Glycosylated Protein." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1274803162.
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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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Mello, Rodrigo Ventura de. "Aplicação de MALDI-TOF MS na caracterização de microalgas da família Selenastraceae (Chlorophyta)." Universidade Federal de São Carlos, 2016. https://repositorio.ufscar.br/handle/ufscar/8748.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
The morphological variability in the Selenastraceae family has been a barrier to a good establishment of well-defined taxonomic groups. Several studies with molecular compounds have already demonstrated its importance in the elucidation of this relation. In this scenario, the present study used a matrix-assisted laser desorption ionization timeof- flight mass-spectrometry (MALDI-TOF MS) technique as a tool to differentiation of the microalgae of this family at the species and strains levels. A group of 18 strains, belong to in 12 different species, of freshwater microalgae was selected. Cells and dissolved organic matter (DOM) were analyzed in the middle exponential growth phase. The analyzes were performed in two different mass ranges: 400 to 2,000 Da and 2,000 to 20,000 Da. Each strain that yielded unique spectra with a good resolution of peaks and reproducibility was selected for clusters analyzes. These spectra were used to make a dissimilarity analysis that showed the capability of differentiation of the strains and species. The strains of the genera Monoraphidium were not all grouped, possibly because it is a polyphyletic group. The praticity and quickness of this technique for data acquisition, allied with the low cost of the analysis, are factors that favor its application in taxonomic studies.
A baixa variedade morfológica presente na família Selenastraceae tem sido uma barreira para o estabelecimento de grupos taxonômicos bem definidos. Estudos realizados com diversos marcadores moleculares já demonstraram a importância dos compostos produzidos por esses organismos na elucidação dessas relações. Nesse cenário, o presente estudo buscou avaliar a aplicação da espectrometria de massa por MALDI-TOF como ferramenta para a discriminação de cepas e espécies dessa família. Foram selecionadas 18 cepas, classificadas em 12 espécies diferentes, de microalgas de água doce. As células e a matéria orgânica dissolvida (MOD) analisadas foram amostradas de cultivos no meio da fase exponencial de crescimento. As análises foram feitas em duas extensões de massa: massas baixas (400 – 2000 Da) e massas altas (2 – 20 kDa). Para cada tipo de análise foram selecionadas as cepas que renderam espectros com boa resolução de picos. Esses espectros foram então utilizados em análises de dissimilaridade para a elaboração de dendrogramas, evidenciando a capacidade da técnica para a distinção de cepas e espécies. Apesar das cepas do gênero Monoraphidium não ficarem todas agrupadas, o que possivelmente ocorre devido ao fato do grupo ser polifilético. A grande rapidez e praticidade desta técnica para a obtenção de dados, aliado ao baixo custo das análises, são fatores que favorecem a sua aplicação neste tipo de estudo.
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Sauerbrey, Kerstin. "Möglichkeiten und Grenzen der Mastitisdiagnostik mittels MALDI-TOF MS-Analytik und molekularbiologischen Methoden." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-179910.
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Olsson, Linnea. "Detection of synergistic activity of antibiotics in Klebsiella pneumoniae using MALDI-TOF MS." Thesis, Örebro universitet, Institutionen för hälsovetenskap och medicin, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-45032.
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