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1
Miller, V. F. "Neuroretinal proteomics". Thesis, Queen's University Belfast, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411748.
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Franchin, Cinzia. "Mass Spectrometry-Based Quantitative Proteomics to Study Proteomes, Phosphoproteomes and Interactomes". Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422169.
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Over the past few years, mass spectrometry-based proteomics has been widely applied to the most diverse fields of biochemistry, biomedicine and biology, and several approaches have been developed to allow absolute and relative quantification of proteins in very complex mixtures. During my PhD, I have conducted three main studies, taking advantage of different quantitative proteomics techniques. In particular SILAC and iTRAQ approaches have been exploited to investigate the calcification process induced by endotoxin in clonal interstitial aortic valve cells, while SILAC and label-free quantitative approaches have been exploited to identify new potential interacting partners and substrates of the protein kinase CK2.
Calcific aortic valve disease represents the most common type of valvular disease and the first cause of surgical valve replacement in the industrialized world. No medical therapies are available to prevent or slow down calcium deposition within the valve leaflets, therefore surgery is the only possible treatment. To investigate the molecular mechanisms underlying the calcification process, SILAC and iTRAQ proteomics approaches have been applied to bovine interstitial aortic valve cells, a cellular model that is able to acquire a pro-calcific profile and drive matrix mineralization upon treatment with an inflammatory stimulus like the endotoxin lipopolysaccharide (LPS). The application to the same cellular model of two different quantitative technologies, led to the identification and relative quantification of hundreds of proteins, among which many showed a significant alteration in response to LPS. The acquired data suggest that cellular oxidoreductase activity, cytoskeletal and spliceosome regulation, glycolisis/gluconeogenesis, and arginine metabolism are altered during the acquisition of the pro-calcific profile. These results represent a starting point to investigate more in detail the molecular mechanisms that seem to be strongly involved in the calcification process induced by LPS.
The other projects described in this thesis focus on CK2, an essential, constitutively active and highly pleiotropic protein kinase. CK2, like many other kinases, is strongly involved in several cellular processes, and in particular it has been hypothesized that this enzyme plays a crucial role in the transduction of survival signals. However, a clear comprehension of the multiple roles played by this kinase within the cell has not been achieved. The aim of these projects was the identification of interacting partners and substrates of CK2 by means of proteomics approaches to try to shed some light on the functions performed by this kinase. In particular a combination of immunoprecipitation experiments and label-free quantitative analyses has been performed to identify new potential interacting partners of CK2, while the SILAC technology, in combination with the use of a specific and potent inhibitor of CK2, was exploited to identify new putative substrates of this kinase directly in a cellular system. The results obtained confirm the notion that CK2 plays a role in many fundamental cellular functions and clearly indicate a strong involvement of this kinase in the biological processes of protein biosynthesis and degradation. Moreover interesting aspects linked to phosphorylation/dephosphorylation turnover rates emerged from these analyses. A detailed discussion, from a technical and biological point of view, of the data collected is presented.
Finally, during my PhD I also collaborated to a project aiming at the identification of the primary molecular targets of antimicrobial photodynamic therapy. This work, not discussed in the thesis, has recently been submitted to the “Journal of Proteomics” with the title: “Molecular Targets of Antimicrobial Photodynamic Therapy Identified by a Proteomic Approach”.Negli ultimi anni, la ricerca proteomica basata sulla spettrometria di massa è stata applicata in modo esponenziale ai più diversi campi della biochimica, biomedicina e biologia, permettendo il parallelo sviluppo di nuovi approcci per la quantificazione relativa e assoluta delle proteine. Nel corso del mio dottorato, ho seguito lo sviluppo di tre progetti principali, sfruttando diverse tecniche di spettrometria quantitativa. In particolare, le tecnologie SILAC e iTRAQ sono state applicate allo studio del processo di calcificazione delle cellule interstiziali delle valvole aortiche, mentre i metodi SILAC e di quantificazione label-free sono stati sfruttati per l’identificazione di potenziali interattori e substrati della protein chinasi CK2. La calcificazione delle valvole aortiche è una delle più comuni patologie valvolari e prima causa di sostituzione valvolare nei paesi industrializzati. A oggi sfortunatamente non esistono terapie che possano prevenire o curare la deposizione di calcio nelle valvole aortiche, e l’unica soluzione è l’intervento chirurgico. Per chiarire le basi molecolari di questo processo, abbiamo applicato le metodiche SILAC e iTRAQ ad un modello cellulare basato su cellule valvolari cardiache bovine (BVIC), in grado di acquisire un profilo pro-calcifico e favorire la mineralizzazione della matrice extra-cellulare in risposta ad uno stimolo infiammatorio come l’endotossina lipopolisaccaride (LPS). L’utilizzo di due diverse tecnologie allo stesso modello cellulare ha permesso l’identificazione, e la relativa quantificazione, di centinaia di proteine, parecchie delle quali mostrano una significativa alterazione in risposta al trattamento con LPS. L’analisi dei dati ha infatti rivelato l’alterazione di proteine appartenenti a diversi processi cellulari, quali la regolazione del citoscheletro, dei meccanismi ossidoriduttivi e dello spliceosoma, la via metabolica della glicolisi/gluconeogenesi, e il metabolismo dell’arginina, suggerendo il coinvolgimento di queste vie nel fenomeno della calcificazione delle valvole aortiche. Questi risultati rappresentano perciò un punto di partenza per nuovi dettagliati studi dei meccanismi molecolari alla base della calcificazione valvolare indotta da LPS. Gli altri progetti descritti in questa tesi sono focalizzato su CK2, una protein chinasi essenziale, altamente pleiotroica e costitutivamente attiva, fortemente implicata in una moltitudine di processi cellulari, in particolare nella trasduzione dei segnali di sopravvivenza, per la quale sembra giocare un ruolo chiave. Tuttavia una completa comprensione del ruolo che CK2 ricopre nei vari processi cellulari in cui è implicata non è ancora stata raggiunta, perciò questo lavoro ha come scopo l’identificazione di nuovi potenziali interattori e substrati di CK2, allo scopo di chiarire maggiormente la sua funzione all’interno della cellula. Nello specifico, abbiamo abbinato esperimenti d’immunoprecipitazione e analisi quantitativa label-free per lo studio delle proteine che interagiscono con CK2, mentre la tecnologia SILAC combinata con l’uso di un inibitore potente e specifico di CK2 è stata applicata alla ricerca di nuovi potenziali substrati di questa chinasi direttamente in un sistema cellulare. I risultati ottenuti confermano le conoscenze già note riguardo al coinvolgimento di CK2 in diversi processi essenziali per la vita cellulare, e fanno emergere chiaramente un coinvolgimento di primo piano di CK2 nei processi di biosintesi e degradazione proteica. Inoltre, l’analisi dei dati ha anche rivelato interessanti ed inattesi aspetti del turnover di fosforilazione/defosforilazione di proteine fosforilate da CK2. I dati ottenuti sono dettagliatamente presentati in questa tesi, da un punto di vista sia tecnico che biologico. Infine, durante il dottorato ho anche collaborato alla realizzazione di un progetto volto all’identificazione di bersagli molecolari nella terapia fotodinamica antimicrobica, utilizzando un approccio proteomico. Da questa collaborazione, è nato un lavoro (non descritto in questa tesi) che è stato recentemente sottoposto a “Journal of Proteomics” con il titolo: “Molecular Targets of Antimicrobial Photodynamic Therapy Identified by a Proteomic Approach”.
3
Walther, Dirk Martin. "Analysis of aging by quantitative proteomics and mitochondrial organellar proteomics". Diss., Ludwig-Maximilians-Universität München, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-174637.
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Ismail, Marcus. "Blodplasmahantering för proteomics". Thesis, Mälardalen University, School of Sustainable Development of Society and Technology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-5485.
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Stone, Helen Marie. "Proteomics in COPD". Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7565/.
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In alpha-1-antitrypsin deficiency (A1ATD) there is excess neutrophil elastase activity, resulting in proteolytic destruction of the lung parenchyma. I hypothesised that the peptide fragments of proteins present in the lung might be detectable in plasma by mass spectrometry and that they might be useful biomarkers of disease activity and treatment efficacy. Calcium ionophore, neutrophil elastase and proteinase 3 were added to plasma from patients with A1ATD to create an in vitro model of the destructive processes. MALDI-based peptide profiling of plasma from patients pre and post treatment with intravenous A1AT was undertaken and MS/MS performed to identify differences. Plasma was also depleted of abundant plasma proteins, labelled with isobaric tags and analysed by shotgun proteomics. The readily detectible components of the plasma proteome remained unchanged with intravenous A1AT. Addition of ionophore, elastase and proteinase 3 to patient blood generated predominantly fragments of fibrinogen. In patients treated with intravenous A1AT, fragments of A1AT increased significantly with treatment: - 2 of these were fragments of a short C-terminal segment of the A1AT protein and were also present in healthy subjects. The shotgun experiments did not identify any robust biomarkers and illustrate the challenging nature of plasma proteomics.
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Le, Thao Thi. "Aptamers for proteomics". Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/1385.
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Changes in post-translation modifications are very important in the regulation of biological processes. Many modifications occur at very low levels, resulting in a low-abundance of the modified proteins in cells, and therefore assessing those modifications is not an easy task. Modern proteomics needs improved methods for identifying such changes. In this thesis, we focus on generating aptamers that can bind phosphoproteins with high affinities and therefore would be able to detect even low-abundance proteins. Aptamers are short sequences of nucleic acids that can be selected from libraries through a process called SELEX to bind targets of interest with high affinity and specificity. In this work, a phosphotyrosine (pY) peptide in a consensus sequence, commonly found in a class of phosphoproteins recognised by SH2 domains of signalling cascades in cells, was chosen as the target. By choosing this peptide target, we aim to create aptamers that can bind a class of proteins that carry this peptide sequence, mimicking the action of the intracellular SH2 domains. An RNA library with 7×1014 molecules with 30 nucleotides in the random region was employed for the selection and aptamers that bind the pY peptide were selected. Using surface plasmon resonance (SPR), binding affinities of these aptamers with their peptide target were determined (Kd values in high nanomolar (nM) range). In addition, aptamers that bind streptavidin tightly (Kd values in low nM range) were also isolated, as streptavidin was used as the matrix in partitioning step during the selection. Affinities of these aptamers were also determined by SPR. Moreover, fluorescence quenching suggested that the streptavidin binding aptamers bound in or near the biotin binding site. These aptamers can be used as affinity tags for RNA molecules. The secondary structures of both types of the aptamers were predicted based on their random-region sequences using the Mfold program.
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Lang, Alastair Michael. "Developing tissue proteomics : differential in gel electrophoresis in biomarker discovery and proteomic degradation". Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4642/.
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The field of proteomics and functional genomics has developed steadily since the completion of the human genome project. The wealth of genomic information and the pace at which it was compiled was astounding. Proteomics, despite considerable effort, on the other hand has not seen quite the same pace of development. The progress being considerably hindered by the lack of an amplification process and the relative complexity of the proteome in comparison to the genome. These intrinsic difficulties have led to the sensitivity of proteomic techniques being pushed closer to physical limits. There is therefore a further need to re-evaluated techniques such as sample preparation and integrity, analytical methods and collaborative strategies to maximise the effectiveness and quality of data collected. The importance of tissue in scientific and clinical research is unequivocal. However, tissue is difficult to collect, store and work with due to issues with proteomic degradation and storage. Good lab practices can minimise the effect of degradation but degradation of proteins can be rapid. Strategies to minimise degradation include freezing, formalin fixing and microwave treatment which all have their relative advantages and disadvantages. The importance of sample preparation as being the top of the workflow is often acknowledged but improvements are not well described in the literature. The main aim of this thesis is to present investigative studies into the mitigation of some of the limitations in tissue sample degradation, analytical approaches in differential in gel electrophoresis and accessing DiGE spot and tissue profile data. Presented is the evaluation of the effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and to aid in the cessation of proteomic degradation. A multifaceted analytical approach of differential in Gel electrophoresis spot data is assessed, giving proteomic profiles of mouse brain tissue. Preliminary data is presented showing that the process of heat-treatment has had a predominantly beneficial effect on mouse brain tissue, with a higher percentage of spots stabilised in heat-treated samples compared to snap-frozen samples. However, stabilisation did occur in snap-frozen samples for different protein spot so the appropriateness of using heat-treatment is as yet not fully determined and requires further analysis. In addition, the variation in tissue profiles of WKY, SP.WKYGla.2a and SHRSP rat model for hypertension is investigated with the future prospect of providing that vital connection between genomic and proteomic data and link phenotype and genotype preliminary investigated. A number of putative markers were identified and quantified using DiGE analysis. In order for these markers to be accepted as biomarkers, more downstream validation is required, however this study provides a good spring board as a proof of concept in using DiGE as an global putative biomarker discovery platform.
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Culwell, Thomas Franklin. "Study of the reproducibility of proteomics methods and variability of fruit fly proteomes /". Diss., CLICK HERE for online access, 2008. http://contentdm.lib.byu.edu/ETD/image/etd2252.pdf.
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Culwell, Thomas Franklin. "Study of the Reproducibility of Proteomics Methods and Variability of Fruit Fly Proteomes". BYU ScholarsArchive, 2007. https://scholarsarchive.byu.edu/etd/1232.
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The reliability of biomarker discovery by means of proteomics has been called into question. It was speculated that "background noise" variation resulting from differences in preparation and handling of samples and proteome dynamics may mask subtle, yet important, differences due to the biological condition. Little is understood about complex proteomes and their variability. A critical aspect of proteomic biomarker research that is largely unexplored is the comparative reproducibility of certain methods such as two-dimensional gel electrophoresis and liquid chromatography/mass spectrometry. In particular, with liquid chromatography/mass spectrometry, it is not known whether variability in peptide quantitation is dependent on any of their several properties such as size, abundance, or hydrophobicity. Such determinations may be critical in properly assessing the value of proteomics data. The fruit fly Drosophila melanogaster was used as a well-controlled multicellular animal model to study the relationship between the background variation and expected changes induced by environmental or genetic factors. The data, gathered by two different proteomics methods, were used to compare and evaluate the reproducibility of the methods. It is reported that there was on average 15 to 18% variability in quantitative measurements of protein abundance using 2-dimensional gel electrophoresis or liquid chromatography/mass spectrometry. Using liquid chromatography/mass spectrometry, peptides with a smaller mass-to-charge ratio were shown to be measured less reproducibly than peptides with a larger ratio. Statistically significant proteomic differences between fly populations could be demonstrated between males and females. In dynamic experiments, less than 0.5% of proteins measured were shown to change after 24 hour starvation of the flies. However, no significant difference in peptide composition could be found for flies fed on a second diet consisting of the standard diet augmented with 10% ethanol. These results suggest that proteomic variability while evident allowed for biomarker discovery using either method for this model system.
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Svensson, Marcus. "Neuropeptidomics expanding proteomics downwards /". Doctoral thesis, Uppsala : Uppsala universitet, Fakultetsövergripande enheter, Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7465.
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Dekker, Leendert Johannes Marinus. "Proteomics of body fluids". [S.l.] : Rotterdam : [The Author] ; Erasmus University [Host], 2007. http://hdl.handle.net/1765/10549.
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Gangadharan, Bevin. "Proteomics in viral disease". Thesis, University of Oxford, 2006. http://ora.ox.ac.uk/objects/uuid:c66c53ed-a824-4f99-8f2b-d2bc65a984c7.
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The separation, identification, and characterisation of the proteins present in a tissue or biological sample is called ‘proteomics’. This technique can be used for example to identify biomarkers and investigate signalling pathways. Increasingly, proteomics is being applied to the analysis of virus related samples; here two such examples are described. Presently there is no reliable non-invasive way of assessing liver fibrosis. Here a novel 2D-PAGE based proteomics study was used to identify potential fibrosis biomarkers. Serum from patients with varying degrees of hepatic scarring induced by infection with the hepatitis C virus (HCV) was analysed. Several proteins associated with liver scarring and/or viral infection were identified. The most prominent changes were observed when comparing serum samples from cirrhotic patients with healthy controls: Expression of inter-α-trypsin inhibitor heavy chain H4 fragments, α1 antichymotrypsin, apolipoprotein L1 (Apo L1), prealbumin and albumin was decreased in cirrhotic serum, whereas CD5 antigen like protein (CD5L) and β2 glycoprotein I (β2GPI) increased. In general, α2 macroglobulin (a2M) and immunoglobulin components increased with hepatic fibrosis whereas haptoglobin and complement components (C3, C4 and factor H-related protein 1) decreased. Novel proteins associated with HCV-induced fibrosis include the inter-alpha-trypsin inhibitor heavy chain H4 fragments, complement factor H-related protein 1, CD5L, Apo L1, β2GPI and the increase in thiolester cleaved products of a2M. The relationship between these changes is discussed. One of the accessory genes of the HIV viral genome encodes for the Nef protein. Nef is present in lipid rafts and increases viral replication within infected host cells by binding to a guanine nucleotide exchange factor, Vav. This leads to activation of a GTPase, Cdc42, however, the signalling pathway is poorly understood. 2D-PAGE based proteomics was used to identify differentially expressed raft-associated proteins by comparing T cells in the presence and absence of Nef. A ubiquitin conjugating enzyme UbcH7, which acts in conjugation with c-Cbl, was absent from the rafts of Nef-transfected cells. Vav ubiquitination was also absent from these rafts. In collaboration with Dr. Alison Simmons and Prof. Andrew McMichael the absence of UbcH7 in rafts was found to be caused by β-Pix forming a ternary complex with c-Cbl and activated Cdc42. Vav ubiquitination was restored and viral replication was diminished when β-Pix was knocked down providing a new candidate target for inhibiting HIV replication. This thesis demonstrates the use of proteomics in providing novel information for virus related samples. This influential technology benefits in both biomarker discovery to aid clinicians with early diagnosis of diseased individuals and in the elucidation of novel signalling pathways in infected cells to provide new candidate targets.
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Xia, Dong. "Proteomics of Toxoplasma gondii". Thesis, University of Liverpool, 2009. http://livrepository.liverpool.ac.uk/1276/.
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The Apicomplexan parasite Toxoplasma gondii is an obligate intracellular parasite. Infection by T .gondii causes the disease toxoplasmosis, which is one of the most prevalent parasitic diseases of animals and humans. It has been 100 years since the first discovery of the parasite in 1908; research on T. gondii has been carried out in many scientific disciplines consistently expanding the understanding of this parasite. In the last ten years, the developments of EST, microarray, genome sequencing and continuing efforts towards genome annotation has centralized the focus of T. gondii research on the understanding of gene expression and gene functions on the genome scale. Equipped with the technical advances in mass spectrometry and bioinformatics, proteomics has become established as an integral component in the post-genomics era by providing first-hand data on the functional products of gene expression. In this study, three complementary proteomic strategies, 1-DE, 2-DE and MudPIT, have been used to characterise the proteome of T. gondii tachyzoites. Protein identifications have been acquired for more than two thousand (2252) unique release 4 genes, representing almost one third (29%) of the predicted proteome of all life cycle stages. Functional predictions for each protein were carried out, which provided valuable insights into the composition of the expressed proteome and their potential biological roles. The T. gondii proteomic data has been integrated into the publically accessible ToxoDB, where 2477 intron-spanning peptides provided supporting evidence for correct splice site annotation of the release 4 genome annotation. The incompleteness of the release 4 genome annotation has been highlighted using peptide evidence, confirming 421 splice sites that are only predicted by alternative gene models. Analysis has also been carried out on the proteomic data in the light of other genome wide expression data. The comparison of the proteome and transcriptome of Toxoplasma and other Apicomplexa parasites has revealed important discrepancies between protein and mRNA expression where interesting candidates have been highlighted for further investigation. A preliminary DIGE study has been developed to characterize protein expression changes in T. gondii grown in the presence or absence of glucose. In conclusion, this study has demonstrated the importance of proteomic applications in understanding gene expression profiles and regulation in T. gondii and highlighted the importance and potential of proteogenomic approaches in genome annotation process. The importance of temporal and quantitative proteomics as well as the future of systems biology has been discussed.
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Ottervald, Jan. "Proteomics in neurological disease". Stockholm, 2009. http://diss.kib.ki.se/2009/978-91-7409-729-0/.
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Niu, Lili. "Escherichia coli proteomics and bioinformatics". [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1240.
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Yaseen, Mumtaz. "Proteomics of Acute Myeloid Leukemia:". Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-69882.
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Michalski, Annette. "Overcoming challenges of shotgun proteomics". Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-151295.
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Champion, Matthew Maurice. "Functional proteomics in Escherichia coli". Texas A&M University, 2005. http://hdl.handle.net/1969.1/3194.
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Cells respond to their environment with programmed changes in gene expression.
Cataloging these changes at the protein level is key towards understanding the physiology
of an organism. Multi-subunit and multi-protein complexes are also important and
pathogenic and physiologic processes. In order to identify expressed proteins and
potential protein complexes, we utilized a combination of non-denaturing
chromatography and peptide mass fingerprinting. This approach allows us to identify the
components of protein mixtures, as well as information lost in traditional proteomics,
such as subunit associations. Applying this methodology to cells at both mid-exponential
and stationary phase growth conditions, we identified several thousand proteins from
each cell-state of E. coli corresponding to hundreds of unique gene products. The copurification
of proteins when fractionated at varying pHs could suggest the components
of higher order complexes. This non-denaturing proteomic approach should provide
physiological data unavailable by other means. The components of several known
cellular complexes were also evident in this analysis. To characterize proteins associated
with nucleic acid binding, we also performed proteome analysis on log and stationary
phase cells grown in LB separated over heparin chromatography at neutral pH, which
enriches for these proteins. The complete analysis of these identifications is discussed.
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Björling, Erik. "Databases for antibody-based proteomics". Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-9658.
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Humans are believed to have ~20,500 protein-coding genes andmuch effort has over the last years been put into the characterizationand localization of the encoded proteins in order to understand theirfunctions. One such effort is the Human Proteome Resource (HPR)project, started in Sweden 2003 with the aim to generate specificantibodies to each human protein and to use those antibodies toanalyze the human proteome by screening human tissues and cells.The work reported in this thesis deals with structuring of data fromantibody-based proteomics assays, with focus on the importance ofaggregating and presenting data in a way that is easy to apprehend.The goals were to model and build databases for collecting, searchingand analyzing data coming out of the large-scale HPR project and tomake all collected data publicly available. A public website, theHuman Protein Atlas, was developed giving all end-users in thescientific community access to the HPR database with proteinexpression data. In 2008, the Human Protein Atlas was released in its4th version containing more than 6000 antibodies, covering more than25% of the human proteins. All the collected protein expression datais searchable on the public website. End-users can query for proteinsthat show high expression in one tissue and no expression in anotherand possibly find tissue specific biomarkers. Queries can also beconstructed to find proteins with different expression levels in normalvs. cancer tissues. The proteins found by such a query could identifypotential biomarkers for cancer that could be used as diagnosticmarkers and maybe even be involved in cancer therapy in the future.Validation of antibodies is important in order to get reliable resultsfrom different assays. It has been noted that some antibodies arereliable in certain assays but not in others and therefore anotherpublicly available database, the Antibodypedia, has been createdwhere any antibody producer can submit their binders together withthe validation data in order for end users to purchase the bestantibody for their protein target and their intended assay.QC 20100708
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Muir, Matthew Stewart. "Proteomics of the ovine cataract". Diss., Lincoln University, 2008. http://hdl.handle.net/10182/792.
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The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.
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Boren, Mats. "Proteomics of barley starch granules /". Uppsala : Dept. of Plantbiology and Forest Genetics, Swedish University of Agricultural Sciences, 2005. http://epsilon.slu.se/2005107.pdf.
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Greninger, Alexander L. "Genomics and Proteomics of Picornaviruses". Thesis, University of California, San Francisco, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3558423.
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Viruses have long been noted to be composed simply of nucleic acid and protein. This thesis describes this confluence of science of viruses at the interface of genomics and proteomics. Chapter 2 describes the discovery of klassevirus, a new picornavirus in pediatric diarrhea. Chapter 3 shows that klassevirus is likely a human pathogen given the seroconversion of klassevirus-positive individuals against a klassevirus non-structural protein that is not present in the picornavirus virion. Subsequent work failed to obtain a culturable virus from klassevirus-positive stool samples, enabling the transition to culture-independent methods of characterizing picornavirus-host protein interactions. Chapter 4 describes the use of affinity purification mass spectrometry to discovery a novel picornavirus 3A-ACBD3-PI4KB complex that promotes viral replication in the enteroviruses and kobuviruses. Chapter 5 extends upon the methodology to describe a novel host protein interactor of ACBD3 (TBC1D22A/B), whose interaction is altered specifically by the kobuvirus 3A protein. This complex also demonstrates significant interaction with the klassevirus 3A protein, suggesting that the AP-MS work may inform the biology of the uncultured virus. Finally, chapter 6 describes future directions that are opened up by this work.
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Bauer, Chris [Verfasser]. "Exploiting proteomics data / Chris Bauer". Berlin : Freie Universität Berlin, 2014. http://d-nb.info/104786231X/34.
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Johnson, Hannah. "New Approaches to Quantitative Proteomics". Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492739.
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Absolute quantitative proteomics typically relies on the use of stable isotope labelled internal standards introduced in a known amount. Comparative signal intensities of the labelled and unlabelled peptides allow the inference of protein concentration.
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Pusch, Miriam. "Proteomics of newly assembled chromatin". Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-180964.
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Volchenboum, Samuel Louis. "Proteomics for cancer biomarker discovery". Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39733.
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Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (p. 51-54).
Background: If we are to successfully treat cancer, we must understand the biologic underpinnings in conjunction with early diagnosis. Genome-wide expression studies have advanced the research of many cancers. Nevertheless, understanding which genes are expressed in a tumor is not equivalent to knowing which proteins are being produced. Proteomics hold great promise for careful examination of the proteins in complex biologic fluids and tissues, and it may be possible to detect disease from a patient's serum, long before it would otherwise be clinically evident. Although there have been steady advances in all the steps of a proteomic analysis, much remains to be standardized. Because of some high-profile problems with the initial analysis of ovarian cancer proteomic data, early exuberance has now been tempered and replaced by a more methodical approach to these studies. Hypothesis: My hypothesis in this thesis is that proteomics is a valuable tool in the diagnosis and study of cancer, as will be demonstrated in several steps. Methods: First, I describe the current field of proteomics, specifically as it applies to early detection of cancer and biomarker discovery.
(cont.) I lay out the current state-of-the-art technologies for preparing samples and enumerating the proteins in complex fluids and tissues, giving special treatment to the main threats to validity-chance and bias. I also describe the bioinformatic tools necessary for analyzing the large amounts of data produced. Through the example of a mouse model of colorectal carcinoma, I demonstrate the steps involved in a proteomic study, from procuring samples to peptide and protein determination to bioinformatic analysis. Finally, I discuss these findings in light of the proteomic considerations discussed earlier. Results: From this work, I discovered that proteomic profiling can describe the proteins in serum from mice both with and without colon cancer. Furthermore, I developed a naive Bayes classifier that could distinguish between the serum of mice with colorectal carcinoma and their normal litter-mates. Contributions: Through this work, I have contributed the following. I described the field of proteomics with special emphasis on cancer biomarker discovery and early detection. I enumerated the challenges and pitfalls to developing early detection schemes for cancer based on high-dimensional proteomic analyses.
(cont.) I described a set of experiments on mice harboring a gene mutation that predisposes them to colorectal carcinoma. I detailed the bioinformatic analysis of this data, including the development of a naive Bayes classifier to differentiate the cancerous state from the normal state. Finally, I discussed the caveats of the current work, in reference to the initial discussion on the challenges and pitfalls of early detection schemes and cancer biomarker discovery.
by Samuel Louis Volchenboum.
S.M.
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Jiang, Yanjie. "Approaches for Improved Positional Proteomics". ScholarWorks@UNO, 2013. http://scholarworks.uno.edu/td/1715.
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Positional proteomics is emerging as an attractive technique to characterize protein termini, which play important biological roles in cells. Even with the advances in past decades, there still are areas for improvement. This thesis focuses on improving data quality and assignment confidence in positional proteomics.
A novel workflow was designed for the large-scale identification of protein N-terminal sequences. 4-sulfophenyl isothiocyanate (SPITC) is used for N-termini sulfonation; Upon higher energy collisional dissociation (HCD), SPITC peptides in electrospray ionization ESI generate predominately y-type ion series; such simplification of spectra enables the identification of N-termini with high fidelity. The presence of b1 + SPITC product ions upon HCD furthers the confidence for N-terminal identifications. Secondly, sulfonated N-terminal peptides possess one negative charge site at low pH, which was exploited to enrich the SPITC modified N-terminal peptides by electrostatic repulsion hydrophilic interaction (ERLIC) chromatography. Such enrichment process allows both N-termini enriched and N-termini deficient fractions to be collected and analyzed by LC-MS/MS. This method was applied to an E. coli cell lysate, identifying approximately 350 N-terminal peptides (85% represented neo-N-termini from protein degradation and 15% from leading methionine excision). These N-terminal peptides represented 274 distinct E.coli proteins, 224 of which were also identified in the analysis of flow-through fractions from internal peptides.
Another approach we took to boost the identification confidence is by exploiting iTRAQ (isobaric tag for relative and absolute quantitation) in the positional proteomics workflow. This approach allows for multiplexed comparison between different samples, and thus is well-suited for degradadomics analyses where degraded samples are compared to control samples. Both control and protease treated sample are labeled by different tags which allows direct comparison of protein N-termini with neo-N-termini. In addition, samples are analyzed duplicate by labeling with two tags, aiming for quick validation of peptides by internal replicates. In this study, Asp-N digested E.coli cell lysate is taken as a model system. A total of 500 N-terminal peptides, corresponding to 370 proteins, were identified with high confidence in one experiment, with 87% of those proteolytic products matching the expected protease digestion specificity, validating the assignment accuracy of this approach.
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Furlan, Cristina. "Quantitative proteomics of human chromatin". Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/17992.
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The work presented in this thesis aims at unravelling human chromatin composition by quantitative proteomics to outline the functional and structural changes occurring during the life of human cells. Chromatin is the structure formed by proteins and RNAs interacting with the genetic material. At present, chromatin is not well defined. It is not easy to investigate either the composition of its constituent proteins or how this arrangement changes. We set out to analyse the chromatin composition changes occurring during the cell cycle. Our procedure couples a SILAC mass spectrometry-based approach with a newly developed biochemical chromatin purification method, which involves fixation of proteins to DNA. By testing two different fixation times (5 and 10 minutes) and three phases of the cell cycle (G1/S, G2, M), we quantified ~3000 proteins providing a broad picture of the global changes on chromatin protein composition. Surprisingly, chromatin seems to be occupied by many unexpected proteins (40%) that appear to be increased during mitosis. We hypothesized that these unexpected proteins come into contact with DNA during mitosis when the nuclear envelope breaks down and the highly negatively charged DNA can be found in proximity to extra nuclear proteins. We used Pulse-SILAC technique that allows to distinguish newly synthesized proteins to test this possibility. By comparing in a single cell cycle and during G0 arrest the incorporation of new proteins into chromatin with their synthesis in the cytoplasm and in the whole cell, we could not find a different behaviour for the unexpected proteins as result of mitosis. Despite the efforts in tracking down the origin of these unexpected proteins, it is still uncertain whether their presence on chromatin is the result of a biological process or, in part, a drawback of the purification methods adopted. However, proving their genuine presence on chromatin will be important to elucidate how chromatin functions.
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Rolland, Catherine. "The proteomics of adipose tissue". Thesis, University of Aberdeen, 2005. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU200569.
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Obesity is a widespread disease both in the UK and worldwide and the prevalence is increasing in epidemic proportions. This increased weight and altered body fat distribution has brought about increased risk of comorbid disease such as type II diabetes, hypertension and cardiovascular disease. The aim of this study was to find novel proteins differentially expressed in subcutaneous and omental adipose tissue. To achieve this, approaches to resolve adipose tissue adequately using two-dimensional electrophoresis (2DE) were developed to allow the detection of differences in protein expression for variables such as fat depot, medication, disease and menses status. Finally, the identification of adipose tissue proteins will allow the development of a much-needed database for human adipose tissue. The analysis of the effects of different variables on protein expression suggested a structural difference as well as an increase in inflammatory markers and an overall greater cellular activity in the omental compared to the subcutaneous adipose tissue depot; an increase in inflammatory markers in the omental adipose depot of obese patients compared to normal weight patients, differences in protein expression in pre- compared to menopausal and post-menopausal patients; and subtle changes in protein expression in adipose tissue in relation to cancer state, although the omental depot displayed a greater number of changes, indicating that endometrial cancer has a greater effect on this depot than the subcutaneous depot. Even at this early stage in the application of 2DE to human adipose tissue, positive results and identification of changes in protein expression in relation to disease and medication have been achieved. With a more extensive use of narrow range IPG gels, protein enrichment methods and fluorescent dyes are promising for the identification and development of a human adipose tissue database and the identification of biomarkers for the detection of disease and development of therapeutic targets.
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Blakeley, Paul. "Computational proteomics for genome annotation". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/computational-proteomics-for-genome-annotation(e48dc673-a8b7-4c78-93ca-aab0eb28ec8a).html.
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The field of proteogenomics operates at the interface between proteomics and genomics, and has emerged during the past decade to exploit the vast quantities of high-throughput sequence data. A range of different proteogenomics approaches have been developed, which integrate mass spectrometry data with genome sequence data to provide empirical evidence for protein-coding genes. However, current methods may not be optimized as they do not fully consider the splicing complexity in eukaryotes and there is currently no best practice method. To address this, we investigate the level of proteomics support for Ensembl gene models in human, and a selection of model organisms. We find a disparity between the number of splice variants confirmed by extant data, and the number that can theoretically be confirmed using current proteomics technologies. We then go on to investigate EST-based proteogenomics methods, which enabled the discovery of novel peptide sequences in the chicken genome, which represent hitherto unannotated genes, amended gene models, polymorphisms, and genes missing from the genome assembly. Different approaches for searching mass spectrometry data against transcript sequences are explored, and we show that searching mass spectra against protein sequences predicted by the EORF and ESTScan2 translation tools results in the best sensitivity.
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Salehi-Reyhani, Ali. "Tools for single cell proteomics". Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/9280.
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Despite recent advances that offer control of single cells, in terms of manipulation and sorting and the ability to measure gene expression, the need to measure protein copy number remains unmet. Measuring protein copy number in single cells and related quantities such as levels of phosphorylation and protein-protein interaction is the basis of single cell proteomics. A technology platform to undertake the analysis of protein copy number from single cells has been developed. The approach described is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by mechanical shearing caused by laser-induced microcavitation; and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. This demonstrates the ability count protein copy number from single cells in a manner which could be applied in principle to any set of proteins and for any cell type without the need for genetic engineering. Metabolism can undergo alteration in diseases such as cancer and heart failure and also as cells differentiate during development. In order to assess how it may inform a proteomic measurement, multidimensional two-photon fluorescence metabolic imaging is conducted on a cultured cancer cell line, primary adult rat cardiomyocytes and human embryonic stem cells. By measuring the parameters of fluorescence such as intensity and lifetime of the autofluorescent metabolic co-factors NADH and FAD, it was found to be possible to contrast cells under various conditions and metabolic stimuli. In particular, human embryonic stem cells were able to be contrasted at 3 stages of development as they underwent differentiation into embryonic stem cell derived cardiomyocytes. Metabolic imaging provides a non-destructive method to monitor cellular metabolic activity with high resolution. This is complimentary to the single cell proteomic platform and the convergence of both techniques holds promise in future investigations into how metabolism influences cell function and the proteome in development and disease.
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Björling, Erik. "Databases for antibody-based proteomics /". Stockholm : Bioteknologi, Kungliga Tekniska högskolan, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-9658.
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Reitz, Nila. "Data mining for phospho-proteomics". Pullman, Wash. : Washington State University, 2009. http://www.dissertations.wsu.edu/Thesis/Fall2009/N_Reitz_120409.pdf.
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Thesis (M.S. in computer science)--Washington State University, December 2009.Title from PDF title page (viewed on Feb. 4, 2010). "School of Electrical Engineering & Computer Science." Includes bibliographical references (p. 49-54).
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Eriksson, Cecilia. "Affinity based proteomics research tools /". Stockholm : Skolan för bioteknologi, Kungliga Tekniska högskolan, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11184.
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Bromilow, Sophie. "Proteomics in 'free-from' foods". Thesis, University of Manchester, 2018. https://www.research.manchester.ac.uk/portal/en/theses/proteomics-in-afreefroma-foods(5793f2a1-9552-46e1-92b4-2fce7fadaa27).html.
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Wheat is the most agronomically important crop with an annual production of approximately 680 million tonnes per year over the five year period of 2008-2012 (Shewry and Tatham 2016). Wheat typically contributes about 20% of the total calorie intake in Western Europe and between 50-70% in some countries in North Africa and in West and Central Asia. It is estimated that in order to meet the continuous growing global demand wheat production needs to increase by 50% by 2050. Wheat is most commonly consumed as bread, pasta and noodles however it is also used as a food ingredient in other types of foods such as sauces and condiments. The versatility of wheat is largely determined by the unique physiochemical properties of gluten (Bailey 1941). Gluten is one of the earliest proteins to be studied, and was first described by Beccari in 1728 (Bailey 1941) and is readily isolated from wheat flour as a viscoelastic mass. Gluten is a complex mixture of proteins which are the major seed storage proteins found in the cereal grains wheat, barley, rye and oats. Gluten accounts for 70-80% of the total protein content in wheat grains and is traditionally divided into two groups based on their solubility called gliadins and glutenins (Osborne 1907). In genetically pre-disposed patients gluten is able to elicit a non-IgE mediated T-cell response known as coeliac disease (CD). CD affects approximately 1% of the global population for which there is no cure. As no cure is available patients must adhere to a strict gluten-free diet which is often costly and socially excluding. The Codex Standard states that gluten-free foods must contain less than 20 ppm of gluten from wheat, barley, rye and oats and their crossbreeds (FAO/WHO 1983). The Codex Standard also recommends using immunobased methods (or alternative methods) that are able to achieve appropriate sensitivity and specificity for the detection and quantification of gluten with a 10 ppm limit of detection (FAO/WHO 1983). Consequently the current gold standard method for detection of gluten is enzyme linked immunosorbent assay (ELISA) utilising the R5 antibody, however this method is not without shortcomings. Proteomics by mass spectrometry has the potential to offer an alternative, complementary method to determine gluten proteins in foods but for the methodology to become fully validated and accepted it must also overcome similar challenges to immunoassay methods, such as effective extraction of samples and the identification of peptide targets with the requisite specificity. In this research a global approach is taken to aid the development of gluten detection methods using mass spectrometry. One of the major hurdles that has stunted the development of mass spectrometry methods for the detection and quantification for gluten is the lack of protein sequence databases which are required to undertake the MS data searching. In the first results chapter of the thesis a curated gluten protein sequence database was developed (GluPro), and investigated for its utility as a MS data searching tool. It was observed that utilising the GluPro database resulted in improved protein identifications. Following the development of the curated database, extensive method development was carried out to undertake the most extensive background characterisation of the gluten proteome to date using discovery proteomics. To ensure the most comprehensive profile was obtained a number of extraction protocols were investigated and two mass spectrometry platforms with intrinsic differences utilising different modes of acquisition were used. This resulted in the most comprehensive profile of the gluten proteome to date being obtained. In order to meet the continually growing global demand for wheat previous mentioned it is considered that this may be done through the use of genetically modified crops with improved traits such as pesticide resistance. Resulting in the very real possibility of GM crops being introduced into the food supply chain, however there is much widespread public concern regarding the toxicity and allergenicity of genetically modified crops. As wheat is already listed as one of the major eight allergens, it is crucial to be able to undertake safety assessments which are able to assess the toxicity and allergenicity and determine if the GM crop is substantially equivalent to the non-GM counterpart. In the third part of this thesis it is shown how the MS method developed in the previous chapters could be applied and has great potential to be used for safety assessment. Further to this, it is demonstrated how utilising the additional information gathered during the curation of the GluPro database was able to ground the results into in silico measure of toxicity. In the final part of this research all information gathered was interrogated to pick appropriate MRM target peptides, which were unique to a single gluten protein and reproducibly observed to be free from modification. The peptides were synthesised with heavy labels to develop a targeted method to replace current ELISA methods for the detection and quantification. Unexpectedly mass shifts were observed for the precursor ion corresponding to deamidation of the synthetic peptides. Further investigation was undertaken to understand the location and cause of the deamidation sites. This development leads into further recommendation for future development of MRM methods for the detection and quantification of gluten.
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Margarucci, Luigi. "Chemical proteomics on ligand protein". Doctoral thesis, Universita degli studi di Salerno, 2011. http://hdl.handle.net/10556/141.
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2009 - 2010The emerging field of mass spectrometry-based chemical proteomics provides a powerful instrument in the target discovery of bioactive small-molecules, such as drugs or natural products[1]. The identification of their macromolecular targets is required for a comprehensive understanding of their bio-pharmacological role and for unraveling their mechanism of action[1, 2]. Indeed, the target discovery of bioactive molecules endowed with intriguing pharmacological profiles is one of the main issues in the field of pharmaceutical sciences, since this is necessary for a rational development of potential drugs. Nevertheless, several bioactive compounds have been mainly evaluated for their pharmacological effects, whereas the exact mechanism of action at molecular level still remains unknown[3, 4]. Moreover, a complementary point of view about the effect of a small bioactive molecule on a cellular system can be given by label-based quantitative proteomic analysis[5]. Indeed, the identification of biologically relevant changes in the expression of proteins in a cell, after a treatment with a bioactive compound, could help to understand the exact mechanism of action of such active compound. Here, we report the application of chemical proteomics to the analysis of the cellular interactome of three marine bioactive metabolites, all showing an intriguing anti-inflammatory pharmacological profile, and the application of quantitative chemical proteomics to the platelets activation mechanism by collagen. In more detail, the chemical proteomic approach was applied to Petrosaspongiolide M (PM)[6-8], Bolinaquinone (BLQ)[9-11] and Perthamide C (PRT)[12] target discovery. Thus, these molecules were immobilized onto agarose beads through an α,ω-diamino polyethylene glycol spacer. The modified beads were then used as baits for fishing the potential partners of the bioactive compounds in macrophages cell lysate. The application of such technique allowed us to identify 20S proteasome, clathrin and endoplasmin (GRP94) as main partners of PM, BLQ and PRT, respectively. Then, in vitro and in cell fluorescence assays were developed to assess the effect of PM onto the 20S proteasome enzymatic system, allowing us to measure the inhibition potency of this sesterterpene on the different proteolytic sites of the proteasome machinery. The BLQ ability to modulate clathrin mediated endocytosis has been assessed through cytofluorimetric and microscopy analysis, suggesting a new application of BLQ as biotechnological tool in the modulation of trafficking pathways. SPR technology has been employed to prove the ability of PRT to interact with GRP94 and Hsp90, opening the way to further investigations on the role of PRT in the modulation of heat shock protein functions. Finally, we report the application of quantitative chemical proteomics to discover the effect of collagen on platelet activation. Since cAMP and cGMP plays a key role in platelet activation[13], we combined quantitative chemical proteomics approach with the specific enrichment of cAMP/cGMP signaling nodes[14], to investigate how PKA but also cGMP-dependent protein kinases (PKG) spatially reorganizes in activated human platelets. [edited by author]
IX n.s.
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Gibson, Frank. "The development of standards for proteomics research and a proteomics investigation of diabetic adipocyte models". Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445541.
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Walther, Dirk Martin [Verfasser], e Matthias [Akademischer Betreuer] Mann. "Analysis of aging by quantitative proteomics and mitochondrial organellar proteomics / Dirk Martin Walther. Betreuer: Matthias Mann". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1058822098/34.
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Pujari, Goutam. "Current and future trends in proteomics (SELDI-TOF) in clinical diagnosis and clinical research". Thesis, Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31972111.
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Razunguzwa, Trust T. "Development of microfluidic devices for proteomics". Morgantown, W. Va. : [West Virginia University Libraries], 2006. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=4691.
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Thesis (Ph. D.)--West Virginia University, 2006.Title from document title page. Document formatted into pages; contains xiv, 131 p. : ill. (some col.). Includes abstract. Includes bibliographical references.
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Handtke, Stefan [Verfasser]. "Proteomics of Bacillus pumilus / Stefan Handtke". Greifswald : Universitätsbibliothek Greifswald, 2016. http://d-nb.info/1088768849/34.
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NAVARRO, JOSÉ FERNANDEZ. "Protein Level Probabilitiesfor Shotgun Proteomics Experiments". Thesis, KTH, Skolan för datavetenskap och kommunikation (CSC), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-137425.
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There exist many techniques to perform the analysis of proteins. Current Shotgun Proteomics based methods assign peptide level scores to peptide-spectrum matches obtained by matching observed spectra against a database of theoretically generated spectra from a set of known proteins. This set of peptide-spectrum matches is ranked according to a score based on the quality of the match. Subsequently, the candidate present proteins can be inferred from the confidently identified peptides. This process seems straightforward and out of the box, however, it has some notable weaknesses. The imperfections of the set of scores given by the database search engine tool lead to the need to apply a post-processing tool that can give more accurate scores yielding more precise information of the number of peptides believed to be present in the sample. However, the set of proteins believed to be present in the analyzed sample should not be inferred directly from the set of high scored peptides. For example, in cases where there are peptides that form part of more than one protein, or when there is a protein that contain both high and low scored peptides, or when there is a high scored peptide that indicates that the protein is present but it is a false positive, etc. Therefore, there is a need for a tool to infer the list of confident proteins from the set of scored peptides and assign confidence scores to the inferred proteins accounting for the problems described. We present in this thesis work a software tool for the analysis of proteins. This tool is based on the integration of the protein inference tool Fido with the postprocessor Percolator as a combined PSM post-processing and protein inference package that efficiently estimates protein level probabilities and confidence scores. We show that our integration of Fido and Percolator surpasses the stateof- the-art protein inference tool Protein prophet in terms of calibration, sensitivity, specificity and number of proteins correctly identified.Proteomik har på senare tid blivit ett mycket viktigt område inom molekylär cellbiologi. Det kan beskrivas som läran om proteiner, hur de modifieras, när och var de uttrycks, hur de är involverade i de metaboliska vägarna samt hur de interagerar med varandra. Det finns många tekniker för analys av proteiner, men "Shotgun Proteomics" är den mest etablerade och anses vara det primära verktyget att studera protiners primärstruktur i stor skala. Nuvarande "Tandem Mass Spectrometry " baserade metoder tilldelar peptidnivåpoäng till peptidspektrumträffar vilka erhålls genom sökning av spektra mot en databas av teoretiskt genererade spectra från kända proteiner. Detta set av peptidspektrumträffar kommer att rangordnas baserat på poäng vilka baseras på kvaliteten på matchningen. Proteinkandidater kan härledas ur ett subset av proteiner identifierade med hög konfidensgrad. Denna process verkar vara simpel, men har dock påvisat uppenbara svagheter vilka vi kommer att beskriva i kommande stycken. Bristerna i poänguppsättningen givna av databasens sökmotor leder till behovet av att applicera ett verktyg för efterbehandling vilket kan ge en mer korrekt poänguppsättning, vilket i sin tur ger mer korrekt information om antalet peptider vilka förväntas finnas i provet. Huvudtanken bakom efterbehandlingsverktyg är att kombinera olika poäng och funktioner givna av sökmotorn för att kunna uppskatta bättre poängrankning av peptider efter deras sannolikhet att befinna sig i provet. Uppsättningen av proteiner vilka tros existera i provet ska dock inte härledas direkt ut ett sett med proteiner med höga poäng. Till exempel, i fall där flera pepetider bildar en del av fler än ett protein, eller när ett protein innehåller peptider med både höga och låga poäng, eller när det finns en peptid med högt poängtal vilket indikerar att proteinet är närvarande, men är egentligen ett falskt positiv, osv. Ett efterbehandlingsverktyg på proteinnivå behövs därför för att tilldela konfidenspoäng till en lista av proteiner vilka tros existera i provet och står för problemet beskrivet ovan.
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Al, Lawati Haider A. J. "Toward a microfluidic system for proteomics". Thesis, University of Hull, 2007. http://hydra.hull.ac.uk/resources/hull:94.
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There has been much interest in the application of microfluidic systems to proteomics in recent years. This is because of the many unique advantages afforded by the reduced dimensions of microfluidic systems compared to classical methods. The reduction in the reaction vessel dimensions leads to a high degree of control, higher sensitivity, better selectivity and reduced analysis time. Initial experimental results have shown the possibility for developing a novel approach for proteomic analysis. A highly efficient protein digestion microdevice was fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected directly to an electrospray mass spectrometer via a ‘microtight T’ connector, from which aqueous acetic acid (0.2 %) was pumped to the mass spectrometer ion source. Six proteins with molecular masses ranging from 2846 to 77703 Da were digested separately, each within eight minutes using this system. In a second set of experiments a short monolithic separation column was fabricated and placed after the immobilized trypsin capillary. This system demonstrated partial separation of the tryptic peptides generated, and detection limits in the pmol range were obtained by utilization of this separation column. The methodology was then transferred to a glass microchip and a novel system was fabricated. The system consists of an on-chip protein digestion channel and on-chip monolithic ion exchange separation column. The digestion system performance was evaluated using single proteins and mixtures of protein. Significant reduction in the digestion time was observed in comparison to the traditional digestion method and a complete digestion was obtained for cytochrome C in less than a minute while a protein mixture was digested within five minutes. The on-chip separation column was initially evaluated using the tryptic digest of cytochrome C. High column efficiency was obtained as indicated from the peak width at half height measurement data (varying between 19.0 to 24.9 s). A detection limit of 3.7 pmol was obtained; lower detection limits may be obtained if a nano-electrospray source were to be used. The separation column was also evaluated by separating a tryptic digest of a mixture of four proteins. Results showed at least 40 % improvement in the sequence coverage due to the separation achieved. In another experiment, the system was successfully used for protein digestion and a first dimension separation followed by an off-line second dimension separation. This was demonstrated using a tryptic digest of BSA and a number of additional peptides were identified as a result of the first dimension separation carried out using the microfluidic system. The system was also tested with a biological sample and initial results gave positive indications; however, this has to be confirmed after optimization of the protein extraction method. The developed system can be used along with the commercially available on-chip reverse phase columns to perform on-chip protein digestion and two dimensional separation of the generated peptides in a high throughput system for the analysis of biological samples. Another novel microfluidic system was also fabricated. The system consists of an on-chip protein digestion channel and on-chip monolithic reverse phase separation column. The microfluidic system was evaluated using cytochrome C, BSA and a mixture of four proteins. The separation and digestion were completed within one hour. However, the column performance was lower than that of the on-chip monolithic ion exchange column previously fabricated as indicated from the peak width at half height measurement data (varying between 30.0 to 115.0 s).
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Falk, Ronny. "Systems enabling antibody-mediated proteomics research". Doctoral thesis, Stockholm, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4025.
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Grossmann, Jonas Tobias. "New strategies in proteomics data analysis". kostenfrei, 2007. http://e-collection.ethbib.ethz.ch/view/eth:29742.
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Elghawanmeh, Omar. "Proteomics characterization of the cardiac sarcolemma". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=99339.
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As the plasma membrane of the cardiac muscle cell (sarcolemma) plays a major role in the cardiac physiology and homeostasis, the aim of this study is to identify using mass spectrometry (MS) and data base mining the integral and peripheral proteins that are making its composition and to uncover putative novel proteins and to ultimately reveal the differences in protein composition between the sarcolemma of control rat hearts and hearts submitted to ischemia and ischemia-reperfusion to find disease-specific markers. Toward this goal we have developed a new method to purify cardiac sarcolemma using an approach that avoids denaturing conditions and combines subcellular fractionation and immunoadsorption: An initial cardiac sarcolemma preparation obtained by sucrose gradient centrifugation was enriched 27-fold in 5-nucleotidase activity and 5-12-fold in sarcolemmal specific markers. Subsequently, this enriched preparation was incubated with antibodies directed against intercalated disc proteins, N-cadherin and Connexin43, and then immunoadsorbed to superparamagnetic beads (Dynabeads Pan Mouse IgG). Samples from the enriched sarcolemma and immunoabsorbed fractions were separated by 1-D SDS-PAGE and in-gel digested with trypsin. The tryptic fragments were identified, analyzed and assigned to proteins using tandem MS-MS spectrometry in combination with Mascot search software. Out of the 518 identified proteins, 40% were attributed to the sarcolemma and associated cytoskeleton, of which 65% were peripheral proteins and the remainders were either membrane spanning or lipid anchored proteins. Further functional classification of the sarcolemma proteins indicates that the majority (65%) are involved in signaling, trafficking and cell adhesion. The MS analysis also reveals the presence of 71 novel proteins, which are currently being evaluated as relevant candidates for further study. Comparing the sarcolemma and immunoselected intercalated disc preparations; we noticed that the distribution of protein abundance in all of the localization and functional categories follows almost the same trend. This led us to conclude that in order to have an easy, fast and a reliable technique to compare different heart states in terms of sarcolemma protein composition the sarcolemma preparation would serve just as well as an immunoselected preparation with less time, effort and materials. In order to find out differences in protein composition between normal sarcolemma and sarcolemma submitted to global ischemia/ischemia-reperfusion; sarcolemma fractions (SF) obtained from Langendorff rat heart models were analyzed by mass spectrometry. The results of this study revealed that global ischemia induced an increase in calcium binding proteins (i.e. annexins) concomitantly with a decrease in transport proteins (i.e. Na/K ATPase). After ischemia, the signaling proteins category showed a decrease followed by a return to the control level after reperfusion. The amount of G alpha inhibiting 2 protein, caveolin, flotilin and myosin varied significantly during the ischemia/reperfusion protocol.
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Khan, Amir Ali. "Proteomics analysis of human stem cells". Thesis, University of Leeds, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.493771.
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Stem cells have two characteristics that make them unique from any other cell: they can renew themselves indefmitely and they can be differentiated into other cell types. The aim of this investigation was to employ proteomic techniques (2D-PAGE/mass spectrometry) to compare protein expression profiles between two embryonic cell lines (HI and Hues1) and between Huesl and a multipotent cell line (MSC).
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Wright, James Christopher. "Techniques to improve Cross-Species Proteomics". Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510938.
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Hudson, Sian Rose. "Towards chemical tagging strategies for proteomics". Thesis, University of York, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.535039.
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Deng, Yi, e 鄧藝. "From neuroimaging to proteomics in schizophrenia". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43278516.
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