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1
Momo, Remi Ako-Mbianyor. "MALDI-ToF mass spectrometry biomarker profiling via multivariate data analysis application in the biopharmaceutical bioprocessing industry." Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/1939.
Full textAbstract:
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) is a technique by which protein profiles can be rapidly produced from biological samples. Proteomic profiling and biomarker identification using MALDI-ToF MS have been utilised widely in microbiology for bacteria identification and in clinical proteomics for disease-related biomarker discovery. To date, the benefits of MALDI-ToF MS have not been realised in the area of mammalian cell culture during bioprocessing. This thesis explores the approach of ‘intact-cell’ MALDI-ToF MS (ICM-MS) combined with projection to latent structures – discriminant analysis (PLS-DA), to discriminate between mammalian cell lines during bioprocessing. Specifically, the industrial collaborator, Lonza Biologics is interested in adopting this approach to discriminate between IgG monoclonal antibody producing Chinese hamster ovaries (CHO) cell lines based on their productivities and identify protein biomarkers which are associated with the cell line productivities. After classifying cell lines into two categories (high/low producers; Hs/Ls), it is hypothesised that Hs and Ls CHO cells exhibit different metabolic profiles and hence differences in phenotypic expression patterns will be observed. The protein expression patterns correlate to the productivities of the cell lines, and introduce between-class variability. The chemometric method of PLS-DA can use this variability to classify the cell lines as Hs or Ls. A number of differentially expressed proteins were matched and identified as biomarkers after a SwissProt/TrEMBL protein database search. The identified proteins revealed that proteins involved in biological processes such as protein biosynthesis, protein folding, glycolysis and cytoskeleton architecture were upregulated in Hs. This study demonstrates that ICM-MS combined with PLS-DA and a protein database search can be a rapid and valuable tool for biomarker discovery in the bioprocessing industry. It may help in providing clues to potential cell genetic engineering targets as well as a tool in process development in the bioprocessing industry. With the completion of the sequencing of the CHO genome, this study provides a foundation for rapid biomarker profiling of CHO cell lines in culture during recombinant protein manufacturing.
2
Rabe, Jan-Hinrich [Verfasser], and Carsten [Akademischer Betreuer] Hopf. "Multimodal FTIR Microscopy-guided Acquisition and Interpretation of MALDI Mass Spectrometry Imaging Data / Jan-Hinrich Rabe ; Betreuer: Carsten Hopf." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1177385341/34.
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3
uk, siricordcc@yahoo co, and Cornelia Charito Siricord. "Detection of Phytophthora species by MALDI-TOF mass spectrometry." Murdoch University, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20070717.125452.
Full textAbstract:
Phytophthora diseases have caused worldwide economic, social and environmental
impacts for decades. Once their presence is confirmed, they are difficult to eradicate. To
reduce and manage the damage inflicted by the pathogen, fast and reliable disease
management protocols are required. Tests that enable the rapid and reliable
identification of the pathogen assist greatly in disease management.
Phytophthora species are traditionally not only detected by baiting but also by plating of
symptomatic tissue on selective media. Species can be identified by the characteristics
of the mycelium growing out of the bait. However, the method is low throughput,
labour intensive, and prone to false negatives. An alternative approach would be to
detect the pathogen by the presence of its DNA. This involves amplification of the
pathogen DNA using Polymerase Chain Reaction (PCR) and detection of the
amplification product. Detection is usually by agarose gel electrophoresis. However,
this is also a labour intensive process involving pouring, loading, running, and staining
of the gels. The aim of this thesis is to explore the use of Matrix Assisted Laser
Desorption/ Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry for detection
of PCR products. This procedure enables the analysis of large numbers of samples
within a very short time-frame as the average time for analysis of each sample is in the
order of milliseconds.
The assay involves annealing an extension (genotyping) primer to the PCR product and
its extension by a single nucleotide. The nature of the nucleotide added differentiates
species as does the site to which the primer anneals. Multiple extension (genotyping)
primers can be used together in a single reaction for detection of multiple species. In
this project four genotyping primers (GPs) were designed from the ITS regions of
Phytophthora palmivora, Phytophthora cinnamomi, Phytophthora citricola, and
Phytophthora cambivora.
The extension primers were tested for their specificity on the DNA of the target species.
The four primers designed were specific for their intended targets except for GPpalm3
which in addition to being extended by ddT when tested with DNA from P. palmivora,
was also extended by ddC when tested with DNA from other species of Phytophthora
or Pythium.
These primers were also tested for their ability to detect multiple Phytophthora species
in a single reaction (multiplexing). Mixtures of primers were added to mixed DNA
templates and the primer extension reaction carried out. The primers were designed so
that their masses were sufficiently different for them to be identified from a mixture.
Six replicates were analysed for each reaction. In general only about 1-3 of the six
replicates gave a positive reaction. This indicates that there may be some interference
between primers, or that the presence of all four nucleotides interfered with the primer
extension reaction. Increasing either the amount of enzyme, the amount of nucleotides
or both did not improve the results.
The sensitivity of detection was tested by the addition of different amounts of mycelium
to soil. The detection sensitivity depended on the primer pair used for PCR
amplification. The ITS1/2 primer pair was more sensitive than the ITS1/4 pair. The
limit of detection was 1 ìg mycelium g soil-1. However using nested PCR, levels of
sensitivity comparable to those obtained using the ITS1/2 primer pair could be
achieved. Primers to other regions of the genome such as the beta cinnamomin elicitin
gene gave very low levels of sensitivity compared to the ITS primers.
In comparison with DNA detection we found that the limit of detection using baiting
was 4 ìg mycelium g soil-1. Results below this limit were unreliable. The method
suffered from the additional disadvantage that it took a long time in comparison to DNA
detection.
DNA detection methods do not distinguish between living and dead organisms in the
soil. However it can be hypothesised that DNA is unlikely to persist for any significant
length of time in soil. To test this, we added plasmid DNA to soil and tested the
persistence of this DNA using a variety of methods such as precipitation of labelled
DNA, southern blotting and PCR amplification. It was found that in general, in soils
from different ecosystems, the bulk of the DNA was undetectable after 24 hours. The
rate of DNA breakdown differed with the soil type. In some soils, the added DNA was
not detected even after 2 hours, whereas in others it could be observed after 10 hours.
The detection depended on the method. Southern blotting showed that although DNA
could be observed at 10 hours, by 24 hours it was completely degraded. In contrast a
PCR product could be obtained from the soil extracts up to 24 hours. In a separate
experiment, plasmid DNA was detectable over a 24 hour incubation period in 5 soil
samples from 5 different sites. The results suggest that DNA is degraded rapidly in soil
and is unlikely to persist longer than 24 hours.
The results in this thesis demonstrate that MALDI-TOF MS is a suitable alternative to
agarose gel electrophoresis for analysis of PCR products. The technique is rapid,
differentiates species from mixtures, is high-throughput and amenable to automation.
Implementation will require further research to automate the primer extension assay to
reduce the sensitivity to impurities in the DNA and to design parameters for sampling
asymptomatic material.
4
Wyatt, Mark Francis. "Analysis of acrylic polymers by MALDI-TOF mass spectrometry." Thesis, Durham University, 2001. http://etheses.dur.ac.uk/3962/.
Full textAbstract:
Poly(methyl methacrylate) (PMMA) homopolymers synthesised using 'classical' anionic methods and subsequently studied by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) are discussed. Specifically, the attempts at different end-group functionalisation reactions, their varying degrees of success, and the characterisation of these functionalized polymers via MALDI are reported. Extra peaks were observed in the spectra of samples containing a tertiary amine end-group. A mechanism for the in situ elimination of H(_2)(g) involving these end-groups, which would fit the observations, is proposed. Two alternative, 'non-classical' routes to the desired materials were investigated, as difficulties in successfully performing capping reactions to give end functionalised PMMA were noted. The first method was a variation of standard anionic polymerisation that involved the use of lithium silanolates, which could be performed at a higher temperature than normal. The second was a controlled free-radical technique known as Reversible Addition-Fragmentation Chain Transfer (RAFT). A lack of control of the polymerisation to the desired degree was observed with the former method. A well-defined RAFT sample was observed to undergo in situ eliminadon also, for which a mechanism involving the dithioester end-group is proposed, and which is supported by MALDI-collision induced dissociation (CID) evidence. The synthesis of block copolymers of various compositions of MMA with r-butyl methacrylate (t-BMA) and hexyl methacrylate (HMA), along with their homopolymers, and their subsequent characterisation is reported. PHMA was analysed easily, in contrast to Pt-BMA. Only copolymers with a high PMMA content were analysed successfully and this has been rationalised in terms of the factors that affect cationisation. The characterisation of equimolar blends of various end-functionalised PMMA samples is reported also. Samples that favour the binding of a metal ion over protonation appear to have a higher ion yield. Once more, these observations are rationalised in terms of the factors that affect cationisation.
5
Siricord, Cornelia Charito. "Detection of Phytophthora species by MALDI-TOF mass spectrometry." Thesis, Siricord, Cornelia Charito (2005) Detection of Phytophthora species by MALDI-TOF mass spectrometry. PhD thesis, Murdoch University, 2005. https://researchrepository.murdoch.edu.au/id/eprint/314/.
Full textAbstract:
Phytophthora diseases have caused worldwide economic, social and environmental impacts for decades. Once their presence is confirmed, they are difficult to eradicate. To reduce and manage the damage inflicted by the pathogen, fast and reliable disease management protocols are required. Tests that enable the rapid and reliable identification of the pathogen assist greatly in disease management.
Phytophthora species are traditionally not only detected by baiting but also by plating of symptomatic tissue on selective media. Species can be identified by the characteristics of the mycelium growing out of the bait. However, the method is low throughput, labour intensive, and prone to false negatives. An alternative approach would be to detect the pathogen by the presence of its DNA. This involves amplification of the pathogen DNA using Polymerase Chain Reaction (PCR) and detection of the amplification product. Detection is usually by agarose gel electrophoresis. However, this is also a labour intensive process involving pouring, loading, running, and staining of the gels. The aim of this thesis is to explore the use of Matrix Assisted Laser Desorption/ Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry for detection of PCR products. This procedure enables the analysis of large numbers of samples within a very short time-frame as the average time for analysis of each sample is in the order of milliseconds.
The assay involves annealing an extension (genotyping) primer to the PCR product and its extension by a single nucleotide. The nature of the nucleotide added differentiates species as does the site to which the primer anneals. Multiple extension (genotyping) primers can be used together in a single reaction for detection of multiple species. In this project four genotyping primers (GPs) were designed from the ITS regions of Phytophthora palmivora, Phytophthora cinnamomi, Phytophthora citricola, and Phytophthora cambivora.
The extension primers were tested for their specificity on the DNA of the target species. The four primers designed were specific for their intended targets except for GPpalm3 which in addition to being extended by ddT when tested with DNA from P. palmivora, was also extended by ddC when tested with DNA from other species of Phytophthora or Pythium.
These primers were also tested for their ability to detect multiple Phytophthora species in a single reaction (multiplexing). Mixtures of primers were added to mixed DNA templates and the primer extension reaction carried out. The primers were designed so that their masses were sufficiently different for them to be identified from a mixture. Six replicates were analysed for each reaction. In general only about 1-3 of the six replicates gave a positive reaction. This indicates that there may be some interference between primers, or that the presence of all four nucleotides interfered with the primer extension reaction. Increasing either the amount of enzyme, the amount of nucleotides or both did not improve the results.
The sensitivity of detection was tested by the addition of different amounts of mycelium to soil. The detection sensitivity depended on the primer pair used for PCR amplification. The ITS1/2 primer pair was more sensitive than the ITS1/4 pair. The limit of detection was 1 mcg mycelium g soil-1. However using nested PCR, levels of sensitivity comparable to those obtained using the ITS1/2 primer pair could be achieved. Primers to other regions of the genome such as the beta cinnamomin elicitin gene gave very low levels of sensitivity compared to the ITS primers.
In comparison with DNA detection we found that the limit of detection using baiting was 4 mcg mycelium g soil-1. Results below this limit were unreliable. The method suffered from the additional disadvantage that it took a long time in comparison to DNA detection.
DNA detection methods do not distinguish between living and dead organisms in the soil. However it can be hypothesised that DNA is unlikely to persist for any significant length of time in soil. To test this, we added plasmid DNA to soil and tested the persistence of this DNA using a variety of methods such as precipitation of labelled DNA, southern blotting and PCR amplification. It was found that in general, in soils from different ecosystems, the bulk of the DNA was undetectable after 24 hours. The rate of DNA breakdown differed with the soil type. In some soils, the added DNA was not detected even after 2 hours, whereas in others it could be observed after 10 hours. The detection depended on the method. Southern blotting showed that although DNA could be observed at 10 hours, by 24 hours it was completely degraded. In contrast a PCR product could be obtained from the soil extracts up to 24 hours. In a separate experiment, plasmid DNA was detectable over a 24 hour incubation period in 5 soil samples from 5 different sites. The results suggest that DNA is degraded rapidly in soil and is unlikely to persist longer than 24 hours.
The results in this thesis demonstrate that MALDI-TOF MS is a suitable alternative to agarose gel electrophoresis for analysis of PCR products. The technique is rapid, differentiates species from mixtures, is high-throughput and amenable to automation. Implementation will require further research to automate the primer extension assay to reduce the sensitivity to impurities in the DNA and to design parameters for sampling asymptomatic material.
6
Siricord, Cornelia Charito. "Detection of Phytophthora species by MALDI-TOF mass spectrometry." Siricord, Cornelia Charito (2005) Detection of Phytophthora species by MALDI-TOF mass spectrometry. PhD thesis, Murdoch University, 2005. http://researchrepository.murdoch.edu.au/314/.
Full textAbstract:
Phytophthora diseases have caused worldwide economic, social and environmental impacts for decades. Once their presence is confirmed, they are difficult to eradicate. To reduce and manage the damage inflicted by the pathogen, fast and reliable disease management protocols are required. Tests that enable the rapid and reliable identification of the pathogen assist greatly in disease management.
Phytophthora species are traditionally not only detected by baiting but also by plating of symptomatic tissue on selective media. Species can be identified by the characteristics of the mycelium growing out of the bait. However, the method is low throughput, labour intensive, and prone to false negatives. An alternative approach would be to detect the pathogen by the presence of its DNA. This involves amplification of the pathogen DNA using Polymerase Chain Reaction (PCR) and detection of the amplification product. Detection is usually by agarose gel electrophoresis. However, this is also a labour intensive process involving pouring, loading, running, and staining of the gels. The aim of this thesis is to explore the use of Matrix Assisted Laser Desorption/ Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry for detection of PCR products. This procedure enables the analysis of large numbers of samples within a very short time-frame as the average time for analysis of each sample is in the order of milliseconds.
The assay involves annealing an extension (genotyping) primer to the PCR product and its extension by a single nucleotide. The nature of the nucleotide added differentiates species as does the site to which the primer anneals. Multiple extension (genotyping) primers can be used together in a single reaction for detection of multiple species. In this project four genotyping primers (GPs) were designed from the ITS regions of Phytophthora palmivora, Phytophthora cinnamomi, Phytophthora citricola, and Phytophthora cambivora.
The extension primers were tested for their specificity on the DNA of the target species. The four primers designed were specific for their intended targets except for GPpalm3 which in addition to being extended by ddT when tested with DNA from P. palmivora, was also extended by ddC when tested with DNA from other species of Phytophthora or Pythium.
These primers were also tested for their ability to detect multiple Phytophthora species in a single reaction (multiplexing). Mixtures of primers were added to mixed DNA templates and the primer extension reaction carried out. The primers were designed so that their masses were sufficiently different for them to be identified from a mixture. Six replicates were analysed for each reaction. In general only about 1-3 of the six replicates gave a positive reaction. This indicates that there may be some interference between primers, or that the presence of all four nucleotides interfered with the primer extension reaction. Increasing either the amount of enzyme, the amount of nucleotides or both did not improve the results.
The sensitivity of detection was tested by the addition of different amounts of mycelium to soil. The detection sensitivity depended on the primer pair used for PCR amplification. The ITS1/2 primer pair was more sensitive than the ITS1/4 pair. The limit of detection was 1 mcg mycelium g soil-1. However using nested PCR, levels of sensitivity comparable to those obtained using the ITS1/2 primer pair could be achieved. Primers to other regions of the genome such as the beta cinnamomin elicitin gene gave very low levels of sensitivity compared to the ITS primers.
In comparison with DNA detection we found that the limit of detection using baiting was 4 mcg mycelium g soil-1. Results below this limit were unreliable. The method suffered from the additional disadvantage that it took a long time in comparison to DNA detection.
DNA detection methods do not distinguish between living and dead organisms in the soil. However it can be hypothesised that DNA is unlikely to persist for any significant length of time in soil. To test this, we added plasmid DNA to soil and tested the persistence of this DNA using a variety of methods such as precipitation of labelled DNA, southern blotting and PCR amplification. It was found that in general, in soils from different ecosystems, the bulk of the DNA was undetectable after 24 hours. The rate of DNA breakdown differed with the soil type. In some soils, the added DNA was not detected even after 2 hours, whereas in others it could be observed after 10 hours. The detection depended on the method. Southern blotting showed that although DNA could be observed at 10 hours, by 24 hours it was completely degraded. In contrast a PCR product could be obtained from the soil extracts up to 24 hours. In a separate experiment, plasmid DNA was detectable over a 24 hour incubation period in 5 soil samples from 5 different sites. The results suggest that DNA is degraded rapidly in soil and is unlikely to persist longer than 24 hours.
The results in this thesis demonstrate that MALDI-TOF MS is a suitable alternative to agarose gel electrophoresis for analysis of PCR products. The technique is rapid, differentiates species from mixtures, is high-throughput and amenable to automation. Implementation will require further research to automate the primer extension assay to reduce the sensitivity to impurities in the DNA and to design parameters for sampling asymptomatic material.
7
Rodrigues, Lívia Riberti 1988. "Análise de impurezas de formas farmacêuticas sólidas por MALDI Mass Spectrometry Imaging (MALDI-MSI)." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312437.
Full textAbstract:
Orientador: Rodrigo Ramos CatharinoTexto em português e inglês
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-25T05:33:49Z (GMT). No. of bitstreams: 1 Rodrigues_LiviaRiberti_M.pdf: 1203619 bytes, checksum: a17baf81fa013532bd6c9d451b2336f2 (MD5) Previous issue date: 2014
Resumo: Atualmente, as doenças cardiovasculares constituem uma das primeiras causas de mortes no Brasil e no mundo. Neste cenário, as estatinas constituem uma notável classe de medicamentos redutores de colesterol e têm sido associadas com uma expressiva diminuição da morbidade e mortalidade cardiovascular para pacientes em prevenção primária ou secundária da doença coronariana. Elas agem inibindo competitivamente a enzima HMG-CoA redutase, através da afinidade destes fármacos pelo sítio ativo da enzima. Esta enzima é responsável por catalisar a conversão do substrato HMG-CoA em mevalonato, um dos precursores do colesterol. A crescente necessidade e busca por medicamentos cada vez mais efetivos traz a preocupação na segurança destes produtos para seus usuários. Neste sentido, o conhecimento das impurezas e produtos de degradação torna-se necessário para garantir sua qualidade. Uma técnica muito utilizada para análises de impurezas e degradantes é a espectrometria de massas, pois é uma técnica sensível e seletiva e permite elucidar as estruturas químicas presentes na formulação do medicamento. Sendo assim, amostras de Atorvastatina cálcica foram analisadas pela técnica de espectrometria de massas por imagem (MALDI-MSI), permitindo a quantificação de impurezas do medicamento através da imagem da distribuição dessa impureza no comprimido. Dessa forma, é possível minimizar o preparo de amostra e obter um melhor conhecimento da formulação
Abstract: Currently, cardiovascular diseases constitute one of the first causes of deaths in Brazil and in the world. In this scenario, the statins are a notable class of medicines and cholesterol reducers have been associated with a significant reduction in cardiovascular morbidity and mortality for patients in primary or secondary prevention of coronary heart disease. They act by inhibiting competitively the enzyme HMG-CoA reductase, through the affinity of these drugs by the active site of the enzyme. This enzyme is responsible for catalyzing the conversion of HMG-CoA to mevalonate substrate, one of the precursors of cholesterol. The growing need and search for increasingly effective drugs brings the concern on the safety of these drugs for their users. In this sense, the knowledge of the impurities and degradation products becomes necessary to ensure their quality. A widely used technique for analysis of impurities and degrading is mass spectrometry, because it is a sensitive and selective technique and allows elucidating the chemical structures of the present formulation of the medicinal product. Thus, samples of Atorvastatin calcium were analyzed by the technique of mass spectrometry imaging (MALDI-MSI), which allows the quantification of impurities from the medicine through the image of the distribution of impurity in the tablet. That way, it is possible minimize sample preparation and get a better understanding of the formulation
Mestrado
Ciencias Biomedicas
Mestra em Ciências Médicas
8
Allwood, Daniel Anthony. "Characterisation and ionisation modelling of matrices in MALDI mass spectrometry." Thesis, University of Hull, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301477.
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Guan, Bing. "Characterization of Oligosaccharides and Nanoparticles by MALDI-TOF Mass Spectrometry." ScholarWorks@UNO, 2007. http://scholarworks.uno.edu/td/585.
Full textAbstract:
The possibilities of differentiating linkage positions and anomeric configurations of small oligosaccharides by negative ion mode MALDI using anion attachment followed by PSD are investigated. By careful initial adjustment of the focusing mirror ratios allowing acquisition of the peaks of interest within the same PSD segment, it is possible to obtain highly reproducible relative ion abundances. Discrimination of different linkage types is achieved by analysis of structurally-informative diagnostic peaks offered by PSD spectra of chloride adducts of oligosaccharides, whereas the relative peak intensities of selected diagnostic fragment pairs make differentiation of anomeric configuration possible. F- and Ac- cannot form anionic adducts with the oligosaccharides in significant yields. However, Br-, I- and NO3- anionic adducts consistently appear in higher abundances relative to [M - H]-, just like Cl-. Mildly acidic saccharides form both deprotonated molecules and anionic adducts, making it possible to simultaneously detect neutral and acidic oligosaccharides via anion attachment. PSD of [oligosaccharide + Cl]- yields structurally-informative fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, whereas PSD of Br-, I- and NO3- adducts of oligosaccharides yield the respective anions as the main product ions without offering structural information concerning the sugar. PSD of the chloride adduct of saccharides containing 1-2 linkages also yields chlorine-containing fragment ions. MALDI-TOF-MS and LDI-TOF-MS are shown to be useful for characterization of ultra-small titania nanoparticles. Peak maxima in MALDI-TOF mass spectra are found to correlate with nanoparticle size. The size distributions of TiO2 nanoparticles, obtained from MALDI- and LDI-TOF-MS are in good agreement with parallel TEM observations. PSD analysis of inorganic x nanomaterials is performed and valuable information about the structure of analytes has been obtained. A group of inorganic nitrate and perchlorate salts of forensic and health interest are investigated by LDI- and MALDI-TOF MS. In each case, a series of characteristic cluster ions are predominant in the negative-ion mode. The number and identity of metal atoms and anions in the recorded cluster ions can be positively identified by their m/z values, distinctive isotopic patterns and characteristic PSD fragmentation patterns.
10
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.
Full textAbstract:
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.
11
Zabet, Moghaddam Masoud. "MALDI mass spectrometry and ionic liquids applications in functional protein analysis /." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=982074360.
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Offei, Felix. "Denoising Tandem Mass Spectrometry Data." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etd/3218.
Full textAbstract:
Protein identification using tandem mass spectrometry (MS/MS) has proven to be an effective way to identify proteins in a biological sample. An observed spectrum is constructed from the data produced by the tandem mass spectrometer. A protein can be identified if the observed spectrum aligns with the theoretical spectrum. However, data generated by the tandem mass spectrometer are affected by errors thus making protein identification challenging in the field of proteomics. Some of these errors include wrong calibration of the instrument, instrument distortion and noise. In this thesis, we present a pre-processing method, which focuses on the removal of noisy data with the hope of aiding in better identification of proteins. We employ the method of binning to reduce the number of noise peaks in the data without sacrificing the alignment of the observed spectrum with the theoretical spectrum. In some cases, the alignment of the two spectra improved.
13
Balluff, Benjamin. "MALDI imaging mass spectrometry in clinical proteomics research of gastric cancer tissues." Diss., lmu, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-155986.
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Djidja, Marie-Claude. "Examination of tumour tissues by direct MALDI-mass spectrometry imaging and profiling." Thesis, Sheffield Hallam University, 2009. http://shura.shu.ac.uk/20662/.
Full textAbstract:
The purpose of the work described in this thesis was to develop and apply efficient methodologies based on MALDI-MSI for the direct analysis and targeting of protein tumour biomarkers within both frozen and formalin fixed paraffin embedded (FFPE) cancerous tissue sections. Method development for protein analysis directly in tumour tissue sections were performed using tumour xenograft models. This involved improvements in sample preparation, such as tissue washing protocols, and the development of data pre-processing methods prior to statistical analysis using a freely available software package, which referred to as Spec Align. The use of MALDI-MSI for studying proteome patterns directly from tumour tissue sections with no requirement for known targets is demonstrated. In addition, in situ identification of proteins within tumour tissue sections was achieved and correlated with their localisation. The method demonstrated here involved the use of octyl glucoside, a non-ionic detergent, which aims to improve the solubilisation and detection of low abundance and membrane-associated proteins within tumour tissue section after on-tissue digestion. The coupling of MALDI-MSI with ion mobility separation (IMS) has been found to improve the specificity and selectivity of the method. Combining these two methodological approaches allowed the targeting and identification of known tumour biomarkers and potential protein markers in various tumour tissue samples including frozen AQ4N dosed colon tumour xenografts and FFPE human adenocarcinoma tissue sections. The localisation and identification of proteins correlated to tumour growth and aggressiveness were studied using IMS-Tag MALDI-MSI, a novel concept developed in this work. In order to demonstrate its use as a potential biomarker discovery tool, MALDI-MSI was used for high throughput analysis of proteins within tissue micro arrays. Combining MALDI-MSI with statistical analysis allowed the design of a novel tumour classification model based on proteomic imaging information after on-tissue digestion. Another challenge for the MALDI-MSI technology is to achieve more targeted quantitative approaches for in situ analysis of proteins. A proof-of-concept based on multiple reaction monitoring (MRM) analysis with MALDI-MSI is described using a high repetition rate solid state laser. This aimed to improve the sensitivity and specificity of the methodology for the investigation of peptides/proteins directly within tumour tissue sections.
15
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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Saraiva, Bruno Manuel Santos. "Identification of bacteria by mass spectrometry." Master's thesis, Universidade de Aveiro, 2014. http://hdl.handle.net/10773/14317.
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Mestrado em Bioquímica - Métodos BiomolecularesIdentification of bacteria is a major part of the diagnosis of a pathogenic infection. In order to positively and confidently identify the bacteria, different techniques can be applied. These techniques are based on different principles, such as phenotypic differences, DNA sequences comparison, mass spectrometry (MALDI-TOF) and the protein content of the bacteria. From comparison of MALDI-TOF for bacteria identification with the other methodologies available, several advantages can be highlighted: lower cost per identification, faster results and higher discrimination power. This work focus on the development of a new application capable of identifying bacteria using MALDI-TOF mass spectrometry. It started by the protein extraction of the samples and the acquisition of the mass profiles of those bacteria. This work proceeded with the development of an application to identify bacteria by comparing the mass profile of an unknown sample with the mass profiles of the bacteria in our database. Using the developed application it was possible to identify both Gram-positive and Gram-negative bacteria to the strain-level. When an identification to strain-level was not possible, it was possible to identify the bacteria to the species-level and in the case the database did not contain an entry of the same species as the sample, the score values were low enough to disregard a possible identification resulting in a low false-positive rate.
A identificação de bactérias é um dos principais componentes do diagnóstico de infeções patogénicas. De modo a se conseguir obter uma identificação podem ser aplicadas diferentes técnicas, tais como, diferenças de fenótipo, comparação de sequências de ADN e a comparação do conteúdo proteico das bactérias. Quando se compara a identificação bacteriana com recurso à espectrometria de massa MALDI-TOF com as metodologias alternativas, podemos destacar diversas vantagens: menor custo por análise, menos tempo para obtenção de resultados e um maior poder discriminatório. Este trabalho tem como foco o desenvolvimento de uma nova aplicação capaz de identificar bactérias com recurso a espectrometria de massa. O trabalho foi iniciado com a extração proteica das amostras e a aquisição dos perfis de massa dessas bactérias. De seguida, prosseguiu-se com o desenvolvimento de uma aplicação para a identificação de bactérias com base na comparação dos perfis de massa da amostra e dos perfis contidos na base de dados. Usando a aplicação desenvolvida conseguiu-se identificar corretamente tanto bactérias Gram-positivas como Gram-negativas. Quando uma identificação da estirpe não foi possível, a aplicação permitiu a identificação da espécie bacteriana e no caso de a base de dados não conter nenhuma entrada que correspondesse a estirpe ou espécie da amostra, os scores obtidos eram suficientemente baixos para uma eventual identificação ser descartada, resultando assim numa baixa taxa de falsos positivos.
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Ben-Farag, Suaad Omran S. "Statistical analysis of mass spectrometry data." Thesis, University of Leeds, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659026.
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The research described in this thesis can be broadly described by term "statistical analysis of mass spectrometry data". Bioinformatics is a new science which attempts to amalgamate statistical methodology with bring statistical thinking and the biological understanding to area which have previously been void of such. Mass spectrometry which is used to study proteins and their functions, is a relatively new field of bioinformatics research. In this thesis we explore three main themes, all of which tackle a different statistical learning method which arises in mass spectrometry. The main focus of the first theme of the research is on using statistical methods to study fragmentation patterns of mass spectrometry experiments. The analysis contained in this theme has been loosely split into parts: firstly, we calculate a probability of a process called cleavage as part of our preliminary analysis to determine which combination of fragmentation site residues were likely to break. In part two, we apply statistical models to investigate factors influencing the relative intensity of fragment ions formed in tandem mass spectrometry experiments. Separate models were formulated for different types of ions as it was thought that different factors may influence the formation of each type of fragment ion. Statistical regression methods are applied to two types of datasets of mass spectra data: tryptic and nontryptic peptide sequences. We find that several factors have a highly significant influence on the relative intensity of fragment ions formed in the experiment.
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Broberg, Susanna. "Studies of oligo- and polysaccharides by MALDI-TOF and ESI-ITMSn mass spectrometry /." Uppsala : Dept. of Chemistry, Swedish Univ. of Agricultural Sciences, 2004. http://epsilon.slu.se/a452.pdf.
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Maurer, Katja. "Oral brush biopsy analysis by MALDI-ToF Mass Spectrometry for early cancer diagnosis." Doctoral thesis, Universitätsbibliothek Leipzig, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-116691.
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Objectives: Intact cell peptidome profiling (ICPP) with MALDI-ToF Mass-Spectrometry holds promise as a non invasive method to detect head and neck squamous cell carcinoma (HNSCC) objectively, which may improve the early diagnosis of oral cancer tremendously. The present study was designed to discriminate between tumour samples and non-cancer controls (healthy mucosa and oral lesions) by analysing complete spectral patterns of intact cells using MALDI-ToF MS.
Material and Methods: In the first step, a data base consisting of 26 patients suffering from HNSCC was established by taking brush biopsy samples of the diseased area and of the healthy buccal mucosa of the respective contralateral area. After performing MALDI-ToF MS on these samples, classification analysis was used as a basis for further classification of the blind study composed of additional 26 samples including HNSCC, oral lesions and healthy mucosa.
Results: By analyzing spectral patterns of the blind study, all cancerous lesions were defined accurately. One incorrect evaluation (false positive) occurred in the lesion cohort, leading to a sensitivity of 100%, a specificity of 93% and an overall accuracy of 96.5%.
Conclusion: ICPP using MALDI-ToF MS is able to distinguish between healthy and cancerous mucosa and between oral lesions and oral cancer with excellent sensitivity and specificity, which may lead to a more impartial early diagnosis of HNSCC.
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Williams, Bradley Allen. "LOCATING CHELERYTHRINE, AN ALKALOID, WITHIN A CYTOSOLIC ENVIRONMENT BY MALDI-TOF MASS SPECTROMETRY." Wright State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=wright1389645518.
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Segu, Mohideen Mohamed Zaneer. "TARGET MODIFICATION FOR ENHANCED PERFORMANCE MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI) MASS SPECTROMETRY." Available to subscribers only, 2008. http://proquest.umi.com/pqdweb?did=1674093101&sid=1&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Thesis (Ph. D.)--Southern Illinois University Carbondale, 2008."Department of Chemistry." Keywords: Enhanced MALDI, MALDI-MS, On-probe separation, Protein-surface interactions, Sublayers, Surface binding capacity. Includes bibliographical references (p. 130-148). Also available online.
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Aboulmagd, Khodier Sarah. "Analysis of Lipids in Kidney Tissue Using High Resolution MALDI Mass Spectrometry Imaging." Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/19443.
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Massenspektrometrisches Imaging (MSI) ist unverzichtbar für die Untersuchung der räumlichen Verteilung von Molekülen in einer Vielzahl von biologischen Proben. Seit seiner Einführung hat sich MALDI zu einer dominierenden Bildgebungsmethode entwickelt, die sich als nützlich erwiesen hat, um die Komplexität von Lipidstrukturen in biologischen Geweben zu bestimmen.
Einerseits ist die Rolle von Cisplatin bei der Behandlung von menschlichen malignen Erkrankungen gut etabliert, jedoch ist Nephrotoxizität eine limitierende Nebenwirkung, die Veränderungen des renalen Lipidprofils beinhaltet. Dies führte zu der Motivation, die Lipidzusammensetzung des Nierengewebes in mit Cisplatin behandelten Ratten zu untersuchen, um die involvierten Lipid-Signalwege aufzuklären. Es wurde eine Methode zur Kartierung der Lipidzusammensetzung in Nierenschnitten unter Verwendung von MALDI MSI entwickelt. Die Verteilung von Nierenlipiden in Cisplatin-behandelten Proben zeigte deutliche Unterschiede in Bezug auf die Kontrollgruppen.
Darüber hinaus wurde die Beurteilung der Ionenbilder von Lipiden in Cisplatin-behandelten Nieren meist als qualitative Aspekte betrachtet. Relative quantitative Vergleiche wurden durch den variablen Einfluss von experimentellen und instrumentellen Bedingungen begrenzt. Daher bestand die Notwendigkeit, ein Normalisierungsverfahren zu entwickeln, das einen Vergleich der Lipidintensität verschiedener Proben ermöglicht. Das Verfahren verwendete einen Tintenstrahldrucker, um eine Mischung der MALDI-Matrix und der internen internen Lipid-Metall-Standards aufzubringen. Unter Verwendung von ICP-MS erlaubte der interne Metallstandard, die Konsistenz der Matrix und der internen Standards zu bestätigen. Die Anwendung der Methode zur Normalisierung von Ionenintensitäten von Nierenlipiden zeigte eine ausgezeichnete Bildkorrektur und ermöglichte einen relativen quantitativen Vergleich von Lipidbildern in Cisplatin-behandelten Proben.Mass spectrometry imaging is indispensable for studying the spatial distribution of molecules within a diverse range of biological samples. Since its introduction, MALDI has become a dominant imaging method, which proved useful to sort out the complexity of lipid structures in biological tissues. The role of cisplatin in the treatment of human malignancies is well-established. However, nephrotoxicity is a limiting side effect that involves an acute injury of the proximal tubule and alterations in the renal lipid profile. This evolved the motivation to study the spatial distribution of lipids in the kidney tissue of cisplatin-treated rats to shed light on the lipid signaling pathways involved. A method for mapping of lipid distributions in kidney sections using MALDI-LTQ-Orbitrap was developed, utilizing the high performance of orbitrap detection. The distribution of kidney lipids in cisplatin-treated samples revealed clear differences with respect to control group, which could be correlated to the proximal tubule injury. The findings highlight the usefulness of MALDI MSI as complementary tool for clinical diagnostics. Furthermore, assessment of the ion images of lipids in cisplatin-treated kidney mostly considered qualitative aspects. Relative quantitative comparisons were limited by the variable influence of experimental and instrumental conditions. Hence, the necessity developed to establish a normalization method allowing comparison of lipid intensity in MALDI imaging measurements of different samples. The method employed an inkjet printer to apply a mixture of the MALDI matrix and dual lipid-metal internal standards. Using ICP-MS, the metal internal standard allowed to confirm the consistency of the matrix and internal standards application. Applying the method to normalize ion intensities of kidney lipids demonstrated excellent image correction and successfully enabled relative quantitative comparison of lipid images in control and cisplatin-treated samples.
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Goolsby, Brian James. "Techniques for improved mass spectrometric analysis of biologically relevant molecules produced by MALDI and ESI in the quadrupole ion trap /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004273.
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Scionti, Vincenzo. "Novel Applications of Mass Spectrometry on Synthetic Polymeric Materials." University of Akron / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=akron1335149657.
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Bowman, Amber Suzanne. "ENHANCED ANALYSIS OF LIGNIN DEHYDROGENATION OLIGOMERS VIA MASS SPECTROMETRY." UKnowledge, 2018. https://uknowledge.uky.edu/chemistry_etds/104.
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Effective analytical techniques need to be developed to characterize the products of lignin degradation experiments to be able to generate renewable products from lignin. Mass spectrometry is an valuable analytical approach for lignin characterizaion, but it is hindered by lignin’s poor ionization efficiency, especially in the positive ion mode. In this work, we attempt to improve lignin’s ionization by utilizing electrospray and laser desorption mass spectrometry coupled with the addition of cations and chemical derivatives. We confronted the ionization problem from both a top-down and bottom-up analytical approach by analyzing synthesized monomers, dimers, and polymers along with natural lignin extracts from switchgrass. We also utilized tandem mass spectrometry to sequence lignin dimers and determine their bonding motifs from their fragmentation patterns. We believe that resolving the ionization issues with lignin will open the door for easier and more efficient lignin break-down techniques and ultimately more accessible renewable products from lignin.
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Oduwole, Elizabeth O. "Generation of a database of mass spectra patterns of selected Mycobacterium species using MALDI-ToF mass spectrometry." Thesis, Stellenbosch : Stellenbosch University, 2008. http://hdl.handle.net/10019.1/2838.
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Thesis (MScMedSc (Pathology. Medical Microbiology))--Stellenbosch University, 2008.The genus Mycobacterium is a group of acid–fast, aerobic, slow- growing organisms which include more than 90 different species. A member of this genus, Mycobacterium tuberculosis, belonging to the Mycobacterium tuberculosis complex (MTB), is the causative agent of tuberculosis (TB). This disease is currently considered a global emergency, with more than 2 million deaths and over 8 million new cases annually. TB is the world’s second most common cause of death after HIV/AIDS. About one-third of the world’s population is estimated to be infected with TB. This catastrophic situation is further compounded by the emergence of Multi Drug Resistant tuberculosis (MDR-TB) and in more recent times, Extensive Drug Resistant tuberculosis (XDR-TB). Early diagnosis is critical to the successful management of patients as it allows informed use of chemotherapy. Also, early diagnosis is also of great importance if the menace of MDR-TB and XDR-TB is to be curbed and controlled. As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Traditional conventional laboratory procedures involving microscopy, culture and sensitivity tests may require turnaround times of 3-4 weeks or longer. Tremendous technological advancement over the years such as the advent of automated liquid culture systems like the BACTEC® 960 and the MGITTM Tube system, and the development of a myriad of molecular techniques most of which involves nucleic acid amplification (NAA) for the rapid identification of mycobacterial isolates from cultures or even directly from clinical specimens have contributed immensely to the early diagnosis of tuberculosis. Most of these NAA tests are nevertheless fraught with various limitations, thus the search for a rapid, sensitive and specific way of diagnosing tuberculosis is still an active area of research. The search has expanded to areas that would otherwise not have been considered ‘conventional’ in diagnostic mycobacteriology. One of such areas is mass spectrometry. This study joins the relatively few studies of its kind encountered in available literature to establish the ground work for the application of mass spectrometry, specifically Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) in the field of diagnostic mycobacteriology. This is an area which is in need of the speed, sensitivity and specificity that MALDI-ToF technique promises to offer. Since this technology is still in its infancy, the use of utmost care in the preparation of reagents, and the handling and storage of the organisms used to generate reference mass spectra for the database cannot be overemphasized. Similarly, the optimization of certain crucial experimental factors such as inactivating method and choice of matrix is of paramount importance. The main aim of this thesis was to generate a database of reference mass spectra fingerprints of selected (repository) Mycobacterium species. This necessitated the standardization of an experimental protocol which ensured that experimental factors and the various instrument parameters were optimized for maximum spectra generation and reproducibility. A standard operating procedure (SOP) for generating the database of reference mass spectra finger print of selected Mycobacterium species was developed and used to investigate the ability of the database to differentiate between species belonging to the same clinical disease complex as well as the nontuberculosis complex. The findings of this study imply that if the defined protocol is followed, the database generated has the potential to routinely identify and differentiate (under experimental conditions) more species of Mycobacterium than is currently practical using PCR and its related techniques. It is therefore a realistic expectation that when the database is clinically validated and tested in the next phase of the study, it will contribute immensely to the diagnosis of tuberculosis and other mycobacterioses. It will also aid in the identification of emerging pathogens particularly amongst the non-tuberculous mycobacteria.
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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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Handley, Kelly. "Statistical analysis of proteomic mass spectrometry data." Thesis, University of Nottingham, 2007. http://eprints.nottingham.ac.uk/10287/.
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This thesis considers the statistical modelling and analysis of proteomic mass spectrometry data. Proteomics is a relatively new field of study and tried and tested methods of analysis do not yet exist. Mass spectrometry output is high-dimensional and so we firstly develop an algorithm to identify peaks in the spectra in order to reduce the dimensionality of the datasets. We use the results along with a variety of classification methods to examine the classification of new spectra based on a training set. Another method to reduce the complexity of the problem is to fit a parametric model to the data. We model the data as a mixture of Gaussian peaks with parameters representing the peak locations, heights and variances, and apply a Bayesian Markov chain Monte Carlo (MCMC) algorithm to obtain their estimates. These resulting estimates are used to identify m/z values where differences are apparent between groups, where the m/z value of an ion is its mass divided by its charge. A multilevel modelling framework is also considered to incorporate the structure in the data and locations exhibiting differences are again obtained. We consider two mass spectrometry datasets in detail. The first consists of mass spectra from breast cancer cells which either have or have not been treated with the chemotherapeutic agent Taxol. The second consists of mass spectra from melanoma cells classified as stage I or stage IV using the TNM system. Using the MCMC and multilevel techniques described above we show that, in both datasets, small subsets of the available m/z values can be identified which exhibit significant differences in protein expression between groups. Also we see that good classification of new data can also be achieved using a small number of m/z values and that the classification rate does not fall greatly when compared with results from the complete spectra. For both datasets we compare our results with those in the literature which use other techniques on the same data. We conclude by discussing potential areas for further research.
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Wandy, Joe. "Unsupervised Bayesian explorations of mass spectrometry data." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/7928/.
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In recent years, the large-scale, untargeted studies of the compounds that serve as workers in the cell (proteins) and the small molecules involved in essential life-sustaining chemical processes (metabolites) have provided insights into a wide array of fields, such as medical diagnostics, drug discovery, personalised medicine and many others. Measurements in such studies are routinely performed using liquid chromatography mass spectrometry (LC-MS) instruments. From these measurements, we obtain a set of peaks having mass-to-charge, retention time (RT) and intensity values. Before further analysis is possible, the raw LC-MS data has to be processed in a data pre-preprocessing pipeline. In the alignment step of the pipeline, peaks from multiple LC-MS measurements have to be matched. In the identification step, the identity of unknown compounds in the sample that generate the observed peaks have to be assigned. Using tandem mass spectrometry, fragmentation peaks characteristic to a compound can be obtained and used to help establish the identity of the compound. Alignment and identification are challenging because the true identities of the entire set of compounds in the sample are unknown, and a single compound can produce many observed peaks, each with a potential drift in its retention time value. These observed peaks are not independent as they can be explained as being generated by the same compound. The aim of this thesis is to introduce methods to group these related peaks and to use these groupings to improve alignment and assist in identification during data pre-processing. Firstly, we introduce a generative model to group related peaks by their retention time. This information is used to influence direct-matching alignment, bringing related peak groups closer during matching. Investigations using benchmark datasets reveal that improved alignment performance is obtained from this approach. Next, we also consider mass information in the grouping process, resulting in PrecursorCluster, a model that performs the grouping of related peaks in metabolomics by their explainable mass relationships, RT and intensity values. Through a second-stage process that matches these related peak groups, peak alignment is produced. Experiments on benchmark datasets show that an improved alignment performance is obtained, while uncertainties in matched peaksets can also be extracted from the method. In the next section, we expand upon this two-stage method and introduce HDPAlign, a model that performs the clustering of related peaks within and across multiple LC-MS runs at once. This allows for matched peaksets and their respective uncertainties to be naturally extracted from the model. Finally, we look at fragmentation peaks used for identification and introduce MS2LDA, a topic model to group related fragmentation features. These groups of related fragmentation features potentially correspond to substructures shared by metabolites and can be used to assist data interpretation during identification. This final section corresponds to a work in progress and points to many interesting avenues for future research.
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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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Friess, Sebastian D. "Surface topology probing of biomolecules unsing noncovalent receptors : A dissetration on MALDI mass spectrometry /." Zürich, 2003. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=15336.
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Heßling, Bernd [Verfasser]. "Complementary application of ESI and MALDI based mass spectrometry in microbial proteomics / Bernd Heßling." Greifswald : Universitätsbibliothek Greifswald, 2013. http://d-nb.info/1032491361/34.
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Watson, R. Craig Jr. "Laser-Ionization Time-of-Flight Mass Spectrometry of High Molecular Mass Inorganic Complexes." Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/35554.
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Laser-Ionization Time-of-Flight Mass Spectrometry (LI-TOF-MS) is a sophisticated tool for the molecular-weight determination and structural characterization of a variety of molecules. Advances in instrumentation and ionization methods have recently expanded its role in the analysis of high-mass analytes. Large multimetallic complexes, which are efficient solar-energy converters, rely heavily on their chemical structure for optimum operation. Molecular mass determinations of these multimetallic complexes have been problematic due to their lability and high molecular weights.
This thesis describes the characterization of a LI-TOF-MS instrument and confirmation of theoretical time-of-flight mass-separation principles. Several test cases demonstrate the instrument's proper operation and calibration for a wide mass range of analytes. Mass spectral results of three organometallic compounds: i. [Ir(dpp)2Cl2](PF6), ii. {[(bpy)2Ru(dpp)]2IrCl2}(PF6)5, and iii. {[(bpy)2Ru(dpp)]2RuCl2}(PF6)5 under a variety of laser ionization and sample preparation conditions are compared. A complete structural characterization of the monometallic complex, [Ir(dpp)2Cl2](PF6), is presented. The two trimetallic analytes fragmented easily, but significant components of the molecules are successfully identified. After optimizing the ionization and analytical procedure, LI-TOF-MS proved useful in the analysis of high molecular mass metal complexes.
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Eastwood, Stephanie. "Development of Amine Containing Polymer Modified MALDI Targets for Complex Peptide and Protein Mixture Fractionation Prior to MALDI Mass Spectrometry Analysis." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/948.
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AN ABSTRACT OF THE DISSERATION OF STEPHANIE EASTWOOD, for the Doctor of Philosophy degree in CHEMISTRY, presented on the DATE OF DEFENSE, at Southern Illinois University Carbondale. TITLE: DEVELOPMENT OF AMINE CONTAINING POLYMERIC MODIFIED MALDI TARGET SUBSTRATES FOR COMPLEX MIXTURE FRACTIONATION OF PEPTIDE AND PROTEIN MIXTURES PRIOR TO MALDI MASS SPECTROMETRY ANALYSIS MAJOR PROFESSOR: Dr. Gary Kinsel The focus of this dissertation is the synthesis, development and application of the amine containing modified targets for the fractionation of complex mixtures of peptides and digested proteins prior to MALDI-MS analysis. Even though MALDI-MS is increasingly used in proteomic applications and analysis, this method suffers from loss of performance in the analysis of mixtures, due to the ion suppression effect. In this effect, basic peptides ionize more readily than acidic peptides and there by suppress the ionization and detection of less basic peptides resulting in the loss in the detection of valuable sequence information and posttranslational modifications. In the approach taken, a modified target containing amine functionality was utilized as a mixture fractionation substrate. The substrates were chosen for the adsorption of targeted analytes. Incorporating this amine function into 3D brushes offer the ability to absorb higher quantities of peptides, allowing for a more thorough analysis and more sequence coverage. In this work fractionation occurs as the result of electrostatic attraction or repulsion between the positively charged polymer surface and negatively charged analytes in solution. Fractionation studies utilizing this amine chemistry for separation were done first with binary peptide mixtures. Basic peptides are observed to repel from the cationic polymer surfaces, allowing the suppressed acidic peptides to separate from the mixture by binding to the amine substrates. The acidic peptides can be eluted and observed. Following successful demonstrations, more complex mixtures of peptides derived from enzymatic tryptic digestion of proteins were studied. Following fractionation, MALDI mass spectra of both the washed and eluted fractions are obtained and ions signals are analyzed and associated with predicted peptide sequences. Complete analysis of the ion signals using Prowl and PeptideMap leads to a percent protein sequence coverage. Comparison of this number with the percent sequence obtained from unfractionated (conventional) digested peptide mixture mass spectra reveals that fractionation does in fact significantly increase and also increases discovery of post translational modifications (PTM). Fractionation studies were studied by varying the pH of tryptically digest proteins. Sequence coverages were found to increase upon fractionation at all pHs studied. In order to increase the discovery of PTMs, the cationic polymer brush was doped with copper ions to selectively capture phosphopeptides. Negatively charged phosphopeptides were shown to be selectively captured by the copper ions complexes within the polymer brush substrate, and subsequently released upon addition of acid.
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Subel, Bethany. "Applications of Mass Spectrometry to Poly(electrolytes) and Kinetics." University of Akron / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=akron1248805724.
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Hunsucker, Stephen Warren. "Time-of-Flight Mass Spectrometry to Characterize Inorganic Coordination Complexes and Cyanobacteria." Diss., Virginia Tech, 2001. http://hdl.handle.net/10919/27150.
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Matrix assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOFMS) is used to study several inorganic coordination complexes and a variety of compounds from cyanobacteria. Also presented is a discussion of TOFMS instrumentation and the improvements in resolution and instrument automation that have lead to widespread and diverse applications of MALDI-TOFMS in all areas of science.
The feasibility of using direct laser desorption/ionization (LDI) TOFMS to detect trace elements in a variety of glass samples using a lithium borate fusion technique for sample preparation is investigated. The result of the fusion technique is a homogeneous incorporation of the analytical sample into a glass. The fusion technique is investigated as a way to eliminate matrix effects in direct LDI-TOFMS analysis. However, the high concentration and low ionization potential of lithium suppress ionization of the elements of interest. The detection limits of elements in glass samples were not at the trace level. Therefore the technique is not as useful as well-established analytical methods like X-ray fluorescence and inductively coupled plasma mass spectrometry.
Direct laser ablation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of four inorganic coordination complexes are discussed. The compounds studied include [Ir(dpp)2Cl2](PF6), {[(bpy)2Ru(dpp)]2RuCl2}(PF6)4, [(tpy)Ru(tpp)Ru(tpp)RhCl3](PF6)4 and {[(bpy)2Ru(dpp)]2IrCl2}(PF6)5 (dpp = 2,3-bis-(2'-pyridyl)-pyrazine, bpy = 2,2'-bipyridine, tpy = 2,2',6',2"-terpyradine, tpp = 2,3,5,6,-tetrakis-(2'-pyridyl)-pyrazine). Spectral intensities and fragmentation patterns are compared and evaluated for several instrument parameters, matrices, and matrix-to-analyte ratios. Direct ablation and MALDI mass spectra of the monometallic complex showed the same ion peaks and differed only in the relative peak intensities. Direct ablation of the trimetallic complexes produced only low-mass fragments containing one metal atom at most. MALDI spectra of the trimetallic complexes exhibited little fragmentation in the high-mass region (>1500 Da) and less fragmentation in the low-mass region compared to direct laser ablation. Proper matrix selection for MALDI analysis was vital, as was an appropriate matrix-to-analyte ratio. The results demonstrate the applicability of MALDI-TOF mass spectrometry for the structural characterization of labile inorganic coordination complexes. A correlation is made between the gas-phase redox chemistry in the MALDI plume and the solution phase electrochemistry for this series of complexes.
MALDI-TOFMS was also used to study compounds isolated from cyanobacteria. A MALDI screening method has been developed to detect the presence of scytonemin, a UV-absorbing pigment. Detection of scytonemin is accomplished by a simple solvent extraction of cyanobacteria in the desiccated state with subsequent MALDI-TOFMS analysis. The method is rapid and semi-quantitative. Cyanobacteria is the only known organism to produce scytonemin, and it is only produced when the organism is subjected to UV stress. Laboratory-grown cultures were subjected to different amounts of UV radiation, and the screening method was used to detect the presence or absence of scytonemin. Cultures grown under ambient conditions (low UV) did not show the presence of scytonemin, while those grown under UV lamps did show the detectable scytonemin. Because scytonemin acts as a biomarker for UV stress, the MALDI screening method could find application in molecular ecology studies of cyanobacteria.
Peptide mass fingerprinting is used to monitor the isolation of the water stress protein from N. commune. The protein is produced by recombinant growth in E. coli in order to assess the role of Wsp in the desiccation tolerance of N. commune. The results show that SDS-PAGE and Western blot analysis are not sufficient to detect the presence of Wsp after purification using ion-exchange chromatography. Three E. coli proteins were identified in the same molecular weight range as Wsp and one of them cross-reacts with the series of antibodies used for the Western blot. The presence of contaminating proteins that cross-react with the immuno assay make it difficult to determine which fractions contained Wsp. Peptide mass fingerprints were obtained for a series of fractions collected after ion-exchange chromatography to pinpoint the location of Wsp. Peptide mass fingerprinting was also used to monitor the stability of the clone and results show that the clone is modified over a six month period.Ph. D.
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Jung, Seokwon. "Surface characterization of biomass by imaging mass spectrometry." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/45906.
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Lignocellulosic biomass (e.g., non food-based agricultural resides and forestry wastes) has recently been promoted for use as a source of bioethanol instead of food-based materials (e.g., corn and sugar cane), however to fully realize these benefits an improved understanding of lignocellulosic recalcitrance must be developed. The primary goal of this thesis is to gain fundamental knowledge about the surface of the plant cell wall, which is to be integrated into understanding biomass recalcitrance. Imaging mass spectrometry by TOF-SIMS and MALDI-IMS is applied to understand detailed spatial and lateral changes of major components in the surface of biomass under submicron scale.
Using TOF-SIMS analysis, we have demonstrated a dilute acid pretreated poplar stem represented chemical differences between surface and bulk compositions. Especially, abundance of xylan was observed on the surface while sugar profile data showed most xylan (ca. 90%) removed from the bulk composition. Water only flowthrough pretreated poplar also represented difference chemistry between surface and bulk, which more cellulose revealed on the surface compared to bulk composition. In order to gain the spatial chemical distribution of biomass, 3-dimensional (3D) analysis of biomass using TOF-SIMS has been firstly introduced in the specific application of understanding recalcitrance. MALDI-IMS was also applied to visualize different molecular weight (e.g., DP) of cellulose oligomers on the surface of biomass.
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Tengstrand, Erik. "Data analysis of non-targeted mass spectrometry experiments." Doctoral thesis, Stockholms universitet, Institutionen för miljövetenskap och analytisk kemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-116820.
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Data processing tools are valuable to the analytical chemist as they can speed up the analysis, and sometimes solve problems that are not feasible to solve in a traditional manner. However, the complexity of many data processing tools can make their use daunting for the inexperienced user. This thesis includes two applications and two tools for data processing. The first application focuses on minimizing the manual input, reducing the time required for a simple task. The second application required more manual input, in the form of parameter selection, but process far more data. The data processing tools both include features that simplify the manual work required. The first by including visual diagnostics tools that helps in setting the parameters. The second via internal validation that makes the tool’s process more robust and reliable, and thereby less sensitive to small changes in the parameters. No matter how good or precise a data processing tool is, if it is so cumbersome that it is not used by the analytical chemists that need it, it is useless. Therefore, the main focus of this thesis is to make data processing easier.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.
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Bauer, Oliver. "Technology development for oligonucleotide fingerprinting applying multiplexed PNA hybridizations and MALDI-TOF mass spectrometry detection /." [S.l. : s.n.], 2003. http://www.diss.fu-berlin.de/2003/326/index.html.
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Gemeinhardt, Chelsea Nicole. "DFT STUDIES OF SMALL ORGANIC MOLECULES FOR APPLICATIONS IN MALDI MASS SPECTROMETRY AND SOLAR CELLS." OpenSIUC, 2019. https://opensiuc.lib.siu.edu/dissertations/1752.
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The focus of the work presented herein is to understand the underlying photoexcitationprocesses of small organic molecules for applications in MALDI mass spectrometry and solarcell applications through computational methods. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TDDFT) calculations were performed to provide insightinto the effect of heavy atom substitution on the charge transfer properties of MALDI matrixmolecules as well as understand the underlying photophysical properties of rationally designedorganic molecules for use in Dye Sensitized Solar Cells (DSSCs) and Organic Solar Cells(OSCs).Fifth and sixth carbon ring substituted isomers of fluorinated, chlorinated, andbrominated isomers of 2,4-dihydroxybenzoic acid and their thermodynamic and photophysicalproperties were examined to understand the effect of halogenation of MALDI matrix molecules.Thermodynamics studies included the calculation of Proton Affinity (PA), Gas Phase Acidity(GPA), and Gas Phase Basicity (GPB). PA values suggested that nearly all halogenated matricesat the ground electronic state became less acidic than its unsubstituted counterpart. Additionally,the GPA values of the unsubstituted isomer revealed that the order of acidity of protons is thecarboxylic acid proton, followed closely by the two phenolic protons. Following fifth ringsubstitution, the carboxylic acid clearly became to most acidic, followed by the second phenolicproton, and lastly the fourth phenolic proton while for the sixth ring substitution, the carboxyliciiacid and second phenolic group yield similar thermodynamic values, but the fourth phenolic group has the most basic GPA value.Comparison of the UV-Vis absorbance spectra generally showed increased absorbance at λmax compared to the unsubstituted isomer, and most importantly, absorbance at MALDI laser excitation wavelength of 337nm increases after halogenation in almost all cases of halogen substitution. An increase in overlap between the absorbance and emission spectra (Stokes shift) occurred after fifth carbon ring halogenation, but not for sixth ring suggesting that the addition of a halogen on the fifth carbon of the aromatic ring results in stokes shiftan overall more rigid molecular structure, while sixth carbon substitution does not.The bond distances, angles, and Mulliken charges for the ground and excited states were tabulated and compared to understand the structural impact of halogenation at two different ring positions, which showed that not only does the exact location of the halogen affect the structure in unique ways, but also the specific identity of the attached halogen.The HOMO-LUMO contours, energies, and gaps were also obtained in the ground, first excited singlet, and triplet states for both sets of isomers. Contours were examined to quantify the role of the halogen as an electron withdrawing group, where the location of the halogen atom, and its identity, leads to different configurations of electron density. The energies of these orbitals are raised or lowered depending on the location of the halogen on the fifth or sixth carbon and whether the molecule is in the ground or excited state.IR spectra were compared for the ground, the first excited singlet, and triplet states to further elucidate the effect of halogenation on the stretching modes of the protons of interest in charge transfer during MALDI. In both fifth and sixth carbon ring substitutions in the groundiiistate, the carboxylic acid proton stretch is not affected while the phenolic protons were to varying degrees.Most interestingly is that, in the singlet state, the second phenolic stretching mode is largely weakened following nearly all halogenation at the fifth and sixth carbon ring position, which may play a role in stabilizing the carboxylate anion left after charge transfer occurs via an intramolecular hydrogen bond. Conversely, in the triplet state, halogenation on the sixth carbon ring position results in a decreased frequency intensity as well as bond strengthening for chlorine and bromine and weakening for fluorine. This in contrast to 5-X-2,4-DHB substitution, where all halogenation weakens the second phenolic group.Short-lived charged states in simple organic donor−acceptor (D−A) systems were also studied as huge improvements are needed to produce efficient photovoltaic devices. One strategy implemented in this work was to prevent back-electron transfer by forming a cascade of energy levels through the use of a donor−acceptor 1−acceptor 2 (D−A1−A2) architecture , where three systems YD-TRC, YD-TRC-AEAQ, and MHTPP-TRC-AEAQ were characterized in depth via computational and experimental methods.The DFT results indicated that YD-TRC could form a D–A1 system, YD-TRC-AEAQ, a D–A1–A2 system, and MHTPP, a D-L-A system. Computational predictions were confirmed via electrochemical measurements included in the Appendix of this work. Ambipolar systems were synthesized, as the HOMO energy of YD and MHTPP were both higher than the HOMO of AEAQ. Furthermore, the red shift exhibited in UV-vis absorption after YD was connected its first acceptor to form YD-TRC was confirmed by computational analysis as an intramolecular charge transfer.ivWhile these studies showed that the addition of a second acceptor extended the lifetime compared to its dyad counterpart, large amounts of energy are still lost during each sequential electron transfer process, as well as the difficulty of synthesizing these molecules in the laboratory. To remedy both concerns, another viable strategy involves designing simple, small Donor-Acceptor (D-A) systems with long charge separation and slow charge recombination.The effect of donor molecules on the compound’s photophysical properties was explored, by selecting multiple donor modules and keeping the accepter the same as used in previous studies. TPA and PCB donors were proposed, and the following compounds were obtained: TPA-TRC and PCB-TRC, respectively. Additionally, dimethoxy groups and dioctyloxy groups were introduced to TPA-TRC, forming the derivatives MeTPA-TRC and OeTPA-TRC, respectively, to be added to this work.In addition to confirming that these compounds are truly arranged in the typical D-A structure based on their orbital energies, we also showed that the HOMO and LUMO are located at the donor module and TRC module, respectively. Most interestingly, the LUMO and LUMO+1 energy levels of these studied systems remained essentially unchanged, while the HOMO energies were impacted, where donor modules with stronger electron donating abilities have stronger effect on the HOMO levels.This work serves to highlight the importance of utilizing computational chemistry throughout the design, screening, and application of small organic dyads and triads toward solar cell applications. As demonstrated, calculations provide insight into the optically active molecular orbitals responsible for electronic transitions, such as those within the UV-Vis range for solar cell applications while the corresponding energies of these same orbitals can be used to accurately screen a potential system for true D-A or D-A1-A2 character.
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Akinapalli, Srikanth. "MICROFLUIDIC DYNAMIC ISOELECTRIC FOCUSING COUPLED TO MATRIX ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY." OpenSIUC, 2016. https://opensiuc.lib.siu.edu/dissertations/1289.
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Proteomics is an increasingly important area of biological research and has gathered much attention over recent years. Major challenges that make a proteomic analysis difficult are sample complexity, diversity and dynamic range. Progress in the area of proteomics relies heavily on new analytical tools for the sensitive, selective, and high-throughput studies of target analytes. It is estimated that there are several hundred thousand proteins in a human cell. In order to be able to analyze such a complex sample, an analytical method must be capable of separating and detecting many different sample peaks. The complexity of such samples indicates that a single separation method will not be able to provide the needed resolution. If two methods that are orthogonal are combined, then the peak capacity of the combined system is the product of the two individual peak capacities. Development of such systems would cater to the current demands of proteomics studies. Matrix assisted laser desorption/ionization (MALDI) mass spectrometry has evolved into a primary analytical tool for proteomics research. MALDI is fast and efficient and has a high tolerance to non-volatile buffers and impurities. The samples for MALDI are typically applied to solid supports after having been subjected to off-line liquid or gel separations. Several methods have been reported involving various chromatographic or electrophoretic separation methods. However, the current methods often require highly sophisticated sample handling systems, which are often expensive and in need of skilled human resources. The current demands of proteomic analyses require fast, efficient and inexpensive methods for separation to fully harness the capability of MALDI mass spectrometry. In this work a microfluidic device has been designed to perform dynamic isoelectric focusing (DIEF) based protein separation with digital sample deposition directly on a MALDI target for offline analysis. DIEF is related to capillary isoelectric focusing which and can facilitate the interface without the loss of the separation resolution. Compared to traditional capillary isoelectric focusing (cIEF) DIEF uses additional high-voltage power supplies to control the pH gradient by manipulating the electric field. The proteins can be focused at a desired sampling position according to their isoelectric point, to be collected for further analysis by MALDI mass spectrometry. DIEF has a peak capacity of over a thousand and offers an ease of interfacing to other techniques making it a preferred separation method for the interface with mass spectrometric techniques such as MALDI. The design of the microfluidic device is based on a digital droplet fractionation. Multiple fractions of the sample solution from DIEF are generated to retain the resolution and to act as an additional separation mode. The microfluidic device is controlled by actuating pneumatic valves built into the device. The DIEF operational parameters were optimized according to the surface functionality and the design of the microfluidic device. A suitable MALDI sample preparation method was found by studying different existing methods. The methods were studied using test proteins prepared in solutions having the additives used in the experiment. A simple mixture of three proteins was used to demonstrate the application of the developed method. The separation between the proteins insulin, hemoglobin and the myoglobin was demonstrated by varying the separation resolution in three experiments.
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Thenstedt, Niklas. "Detektion av hydrolyserad β-laktamantibiotika i plasma med Matrix-Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry och Liquid Chromatography tandem Mass Spectrometry." Thesis, Örebro universitet, Institutionen för hälsovetenskaper, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-84594.
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Introduktion Antibiotikaresistens är ett globalt växande problem. Till gruppen β-laktamantibiotika hör piperacillin-tazobaktam och cefotaxim som båda verkar genom att försvaga cellväggen med kovalenta bindningar till peptidoglykanlagret som lyserar cellen. E. coli och K. pneumoniae tillhör gruppen Enterobacteriaceae, som är en del av den humana tarmfloran och ofta förekommande vid urinvägsinfektion och sepsis. Utvidgat Spektrum β-Laktamas (ESBL) är ett enzym som finns hos Enterobacteriaceae och som hydrolyserar β-laktamantibiotika. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) är en kvalitativ analysteknik för detektion av kemiska föreningar i avseende på massa och laddning. Kännedom om antibiotikametaboliters molekylvikt vid hydrolys möjliggör detektion. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) är en högsensitiv kvantifieringsmetod som separerar molekyler i avseende på polaritet för vidare detektion i avseende på massa och laddning. Syfte Syftet med denna studie var att vidareutveckla en snabb och effektiv metod för att påvisa nedbrytning av piperacillin-tazobaktam och cefotaxim i blodplasma med LC-MS/MS. Material och Metod Tiofaldigt sjunkande koncentrationer av piperacillin-tazobaktam från 2000 till 2 µg/ml, och cefotaxim med koncentrationerna 500 till 0,5 µg/ml analyserades med MALDI-TOF MS, dels intakt men även med bakterierna E. coli och K. pneumoniae med uttryck av olika resistensmekanismer. Vid optimerade koncentrationer spikades plasmaprover med nedbrutet antibiotika som sedan kvantifierades med LC-MS/MS. Resultat Lägsta detektionsgräns med MALDI-TOF MS för intakt och hydrolyserat piperacillin-tazobaktam var 20/2,5 µg/ml. För cefotaxim var lägsta gränsen 5 µg/ml. Med kliniskt relevanta blodkoncentrationer gick hydrolys inte att detektera för. Med tre bakteriekolonier/50 µl kunde dock hydrolys detekteras och kvantifieras med LC-MS/MS. Slutsats Detektion av β-laktamantibiotika är möjligt med både MALDI-TOF MS och LC-MS/MS. För att påvisa hydrolys krävdes större mängder bakterier än förväntat med LC-MS/MS.Introduction Antibiotic resistance is a global growing problem. Piperacillin-tazobactam and cefotaxime are parts of the group β-lactam antibiotics. The common feature is to inhibit the cell wall synthesis by covalent bindings to the peptidoglycan layer and thereby causing lysis of the bacterial cell. E. coli and K. pneumoniae are members of the Enterobacteriaceae which is a part of the human normal flora but also are commonly associated with urinary tract infections which sometimes develops into to sepsis. Extended Spectrum β-Lactamases (ESBLs) are enzymes with hydrolytic abilities acting on β-lactam antibiotics, expressed by Enterobacteriaceae. The qualitative, Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to detect chemical compounds in the ratio of mass to charge in accordance to their molecular weight. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) is a highly sensitive two-step method of quantification which first separate molecules by their polarity attraction force and then by the ratio of mass to charge. Aim The aim of this study was to develop a fast and efficient method to determine degradation of piperacillin-tazobactam and cefotaxime in blood plasma by LC-MS/MS. Method Tenfold dilution of piperacillin-tazobactam in concentrations of 2000 to 2 µg/ml, and cefotaxime in concentrations of 500 to 0,5 µg/ml where analyzed by MALDI-TOF MS, intact and also with the bacteria E. coli and K. pneumoniae with different expression of antibiotic resistance. Optimized concentrations where fixed in blood plasma and then quantified by LC-MS/MS. Result The detection limit by using MALDI TOF MS of hydrolyzed as well as non-hydrolyzed piperacillin-tazobactam was 20/2,5 µg/ml. The detection limit in cefotaxime was 5 µg/ml. Hydrolysis could not be detected in clinically fixed blood concentrations. Detection and quantification of hydrolysis by LC-MS/MS was possible in a concentration of three bacteria colonies/50 µl. Conclusion It is possible to detect hydrolysis in both MALDI TOF MS and LC-MS/MS. A larger amount of bacteria than expected was needed to demonstrate hydrolysis In LC-MS/MS.
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Djidja, M.-C., V. A. Carolan, Paul M. Loadman, and M. R. Clench. "Method development for protein profiling in biological tissues by matrix-assisted laser desorption/ionisation mass spectrometry imaging." Wiley, 2008. http://hdl.handle.net/10454/4568.
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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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Hettick, Justin Michael. "Optimization and utilization of MALDI 193-nm photofragment time-of-flight mass spectrometry for peptide sequencing." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/1197.
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This study focuses on the application of 193-nm excimer laser (ArF) photodissociation to tandem time-of-flight mass spectrometry. In particular, it focuses on identifying the optimal experimental conditions for peptide sequencing and applying the technology to interesting systems. The early focus is on optimizing the sample preparation conditions that define the initial internal energy state of MALDI-produced ions. Subsequent chapters investigate the effect of changing photodissociation laser conditions and define conditions under which the information content of the spectrum is maximized. Later chapters compare the photodissociation experiment to technologies that represent the current state of the art in tandem mass spectrometry, illustrating both the advantages and shortcoming of photodissociation TOF methodology. Finally, we apply photodissociation to the study of interesting systems of biological relevance, including (1) peptides derived from enzymatic digestion, (2) post-translationally modified peptides, and (3) peptide-transition metal ion complexes. In the final chapter we consider the analytical implications of the work as a whole and comment on the analytical viability of the methodology and look forward to new directions for the experiments.
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Woldegiorgis, Andreas. "Fabrication of new silica nano structures and development of new concepts for MALDI-TOF mass spectrometry." Doctoral thesis, Stockholm : Tekniska högsk, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-79.
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Jia, Wei Jie. "The application of MALDI and tandem reflectron TOF mass spectrometry to the analysis of biomolecular ions." Thesis, University of Glasgow, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321839.
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Reeves, Savanah Gail. "Condensed tannin characterization with FT-ICR MALDI mass spectrometry and separation with saw-tooth gradient HPLC." Miami University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=miami1591185101154831.
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Walton, Barbara Lynn. "A Study of Silver: an Alternative Maldi Matrix for Low Weight Compounds and Mass Spectrometry Imaging." Thesis, University of North Texas, 2014. https://digital.library.unt.edu/ark:/67531/metadc499981/.
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Soft-landing ion mobility has applicability in a variety of areas. The ability to produce material and collect a sufficient amount for further analysis and applications is the key goal of this technique. Soft-landing ion mobility has provided a way to deposit material in a controllable fashion, and can be tailored to specific applications. Changing the conditions at which soft-landing ion mobility occurs effects the characteristics of the resulting particles (size, distribution/coverage on the surface). Longer deposition times generated more material on the surface; however, higher pressures increased material loss due to diffusion. Larger particles were landed when using higher pressures, and increased laser energy at ablation. The utilization of this technique for the deposition of silver clusters has provided a solvent free matrix application technique for MALDI-MS. The low kinetic energy of incident ions along with the solvent free nature of soft-landing ion mobility lead to a technique capable of imaging sensitive samples and low mass analysis. The lack of significant interference as seen by traditional organic matrices is avoided with the use of metallic particles, providing a major enhancement in the ability to analyze low mass compounds by MALDI.
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Lee, Wooram. "Protein Set for Normalization of Quantitative Mass Spectrometry Data." Thesis, Virginia Tech, 2014. http://hdl.handle.net/10919/54554.
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Mass spectrometry has been recognized as a prominent analytical technique for peptide and protein identification and quantitation. With the advent of soft ionization methods, such as electrospray ionization and matrix assisted laser desorption/ionization, mass spectrometry has opened a new era for protein and proteome analysis. Due to its high-throughput and high-resolution character, along with the development of powerful data analysis software tools, mass spectrometry has become the most popular method for quantitative proteomics.
Stable isotope labeling and label-free quantitation methods are widely used in quantitative mass spectrometry experiments. Proteins with stable expression level and key roles in basic cellular functions such as actin, tubulin and glyceraldehyde-3-phosphate dehydrogenase, are frequently utilized as internal controls in biological experiments. However, recent studies have shown that the expression level of such commonly used housekeeping proteins is dependent on cell type, cell cycle or disease status, and that it can change as a result of a biochemical stimulation. Such phenomena can, therefore, substantially compromise the use of these proteins for data validation.
In this work, we propose a novel set of proteins for quantitative mass spectrometry that can be used either for data normalization or validation purposes. The protein set was generated from cell cycle experiments performed with MCF-7, an estrogen receptor positive breast cancer cell line, and MCF-10A, a non-tumorigenic immortalized breast cell line. The protein set was selected from a list of 3700 proteins identified in the different cellular sub-fractions and cell cycle stages of MCF-7/MCF-10A cells, based on the stability of spectral count data (CV<30 %) generated with an LTQ ion trap mass spectrometer. A total of 34 proteins qualified as endogenous standards for the nuclear, and 75 for the cytoplasmic cell fractions, respectively. The validation of these proteins was performed with a complementary, Her2+, SKBR-3 cell line. Based on the outcome of these experiments, it is anticipated that the proposed protein set will find applicability for data normalization/validation in a broader range of mechanistic biological studies that involve the use of cell lines.Master of Science