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Ciryam, Prajwal. "Proteome metastability in stress, aging, and disease." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708160.
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McDonald, Lucy. "Positional proteomics : advanced strategies for targeted proteome simplification." Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501607.
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Proteome complexity presents a major challenge in the field of proteomics. The majority of bottom-up methods begin with proteolysis, which increases the number of analytes in the mixture by about 30-50 fold. This level of complexity demands simplification, and there is an increasing requirement for strategies and reagents that reduce the complexity of a total proteome mixture. It may be argued that when analysing a complete protein digest, for instance by standard shotgun methods, more peptides are analysed than strictly necessary. An efficient proteomic strategy simplifies the proteome while preserving most of the information necessary for comprehensive analysis. A practical approach to proteome simplification is to target a specific structural region of the protein molecule. The ultimate simplification strategy would be to select a single signature peptide from each protein in the proteome.
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Sun, Jin. "Characterization of the egg and embryonic proteome of Pomacea canaliculata, and responses of the proteome to environmental stressors." HKBU Institutional Repository, 2013. http://repository.hkbu.edu.hk/etd_ra/1519.
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Woolerton, Yvonne. "Quantitative proteomics strategies to explore the Saccharomyces cerevisiae proteome." Thesis, University of Liverpool, 2014. http://livrepository.liverpool.ac.uk/2025519/.
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Quantitative proteomics aims at not just identifying, but accurately quantifying the cellular proteome, and while technological advances towards accurate and reliable quantification of proteins is advancing, this alone does not provide an accurate picture of a proteins role within a cell. There is a far greater level of functionality in the cellular environment than there are protein coding genes in the genome, owing partly to the organisation of individual proteins into larger assemblies. A single protein can form interactions with, potentially, a large number of other proteins, leading to a variety of different protein complexes, and subunits can move, break apart or combine depending on cellular conditions. This complex organisation is despite the normal proteomics strategies employing a destructive process, breaking protein structure down to the peptide level. Further difficulties in mapping the cellular proteome arise from the differential expression level of proteins, which in S.cerevisiae can span up to 5 orders of magnitude. This poses problems for the quantification of less abundant proteins in the cell, which can be masked by the more concentrated proteins. An attempt is made within this thesis to use quantitative proteomic techniques to build a picture of the S.cerevisiae cellular proteome. For the analysis of S.cerevisiae protein complexes ion exchange chromatography has been used to separate the cellular proteome into discrete fractions, each containing a different array of protein complexes. The aim here was to analyse the individual subunits of these complexes by LC-MS, with the use of label free quantification strategies. This enables the high throughput identification and quantification of 1800 proteins along with their potential interaction partners. However, for some of the complexes presented here the accuracy of the label free quantification is called into question, as complex subunits known to be equimolar are identified at different concentrations. In order to assess the accuracy of the label free data QconCATs were also designed to analyse the subunits of some complexes by label mediated quantification. In addition, an attempt is made to access proteins from the entire dynamic range of the cellular proteome using equaliser bead technology. This method uses a library of hexapeptide ligands bound to porous beads to bind, theoretically, every protein present in the sample to equal amounts. The beads are used here to bring up the less abundant proteins in the sample, while simultaneously reducing the amount of the abundant proteins. While this goal is achieved, it is also evident that certain proteins are able to bind the beads to a much larger extent than others, so rather than reducing the dynamic range of proteins identified, there is more of a shift in the dynamics, with previously mid-range proteins becoming highly abundant in the data presented here.
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Klammer, Aaron A. "Revealing the proteome : a machine learning approach to peptide identification /." Thesis, Connect to this title online; UW restricted, 2008. http://hdl.handle.net/1773/10278.
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Branson, Owen E. "Improved tag-count approaches for label-free quantitation of proteome differences in bottom-up proteomic experiments." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1471553685.
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Yee, Fong-ying Anita, and 伊芳盈. "Transcriptome and proteome of the intervertebral disc in health and disease." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B43752354.
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Yee, Fong-ying Anita. "Transcriptome and proteome of the intervertebral disc in health and disease." Click to view the E-thesis via HKUTO, 2010. http://sunzi.lib.hku.hk/hkuto/record/B43752354.
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Fisher, Christal. "Quantitative analysis of the plasma proteome in pre-eclampsia." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/quantitative-analysis-of-the-plasma-proteome-in-preeclampsia(3e207341-ebb9-4cb0-b7ea-34b9b110eda6).html.
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There is currently no clinically useful screening test available to identify nulliparous women at high risk of developing pre-eclampsia. This study aimed to identify novel biomarkers using hypothesis generating proteomic methods applied to plasma samples obtained prior to clinical diagnosis of pre-eclampsia. Plasma samples taken at 15 weeks gestation from women who subsequently developed late pre-eclampsia (> 34 weeks), early pre-eclampsia (< 34 weeks) and two distinct groups of women with uncomplicated pregnancies (each n=12) were pooled. Pooled plasma was immunodepleted, labelled using iTRAQ-8 plex reagent and separated into fractions using high pH reverse phase chromatography. Fractions were analysed by LC-MS/MS and data interrogated using ProteinPilot 3.0. The merits of two immunodepletion systems were compared; the Seppro® IgY 14 -SuperMix LC column system removes up to 100 highly abundant plasma proteins and the Multiple Affinity Removal LC column depletes 14 highly abundant plasma proteins. Removal of more high abundance proteins allowed identification of more, potentially interesting, low abundance proteins, but was less reproducible than removing fewer proteins. Two methods of LC-MS/MS analysis were assessed; the QStar XL qTOF and 5800 MALDI-TOF-TOF. The protein identifications and the quantification data acquired by each method was comparable and complementary and increased the total number of proteins identified. A total of 502 proteins were identified. A stringent two stage analysis was developed to identify candidate proteins which changed in abundance in plasma from women who later developed pre-eclampsia compared to women with uncomplicated pregnancies. Analysis identified a total of 113 proteins which were both reproducibly quantified and changed by more than the expected range of biological variation. Six candidate proteins changed in abundance in the plasma taken from women who subsequently developed early pre-eclampsia were selected for further validation. A high throughput, low cost, method of multiple reaction monitoring which allows relative quantitation without the use of costly isotopically labelled peptides was developed to validate candidate proteins. Candidate proteins were also assessed by western blot and ELISA. Only one candidate protein; platelet basic protein, was validated by all three methods and demonstrated similar increases in the abundance. This investigation suggests that measurement of platelet basic protein at 15 weeks gestation is a novel candidate predictive marker for pre-eclampsia. Validation of platelet basic protein in a large, independent, sample set is required to confirm changes in protein expression and to evaluate potential, alongside other factors, to identify nulliparous women at high risk of developing pre-eclampsia later in pregnancy.
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Tabb, David L. "Bioinformatics of proteomic tandem mass spectra : selection, characterization, and identification /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/10847.
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Ozgazi, Nese. "Proteome Analysis Of Blumeria Graminis F. Sp. Hordei Inoculated Barley." Master's thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/3/12611152/index.pdf.
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Blumeria graminis f. sp. hordei is a biotroph pathogen that causes powdery mildew disease in barley. In this study, Pallas01 and Pallas03 barley lines having Mla1, Ml (Al2) and Mla6, Mla14 R-genes were inoculated with Bgh103(64/01) race of the Blumeria graminis f. sp. hordei having avirulence and virulence to Pallas01 and Pallas03, respectively.
The proteins were isolated from the three biological replicates of 12, 24, and 48 hpi samples following the method in Rampitsch et al., 2006. These there biological replicates of three time points together with the mock inoculated plant proteins were separated on 2D-PAGE using IPG strips of 4-7 pH values as three technical replicates, resulting 108 gels.
The gels were analyzed using PdQuest (Bio Rad) in order to assess up- or down-regulated protein spots by comparing against controls and the samples having resistance or susceptible responses with each other. According to the analysis, 36 proteins were found to be differentiated and among them 18 proteins were found up-regulated and 8 proteins were found down-regulated. The spots were manually
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excised and subjected to the nano-LC-ESI-MS/MS analysis (Proteome Factory, Germany). The MASCOT algorithm was used for identification of the possible proteins. The experimental pI and MW values were used for selecting the differentiated proteins from the mass results.
The relative abundance of each of the 38 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be carbohydrate metabolism related. The relative distribution of the proteins into four main functional categories was taken into consideration.
Statistical tests (Students‟
T-test) were carried among the identified proteins in order to reveal statistically significant proteins throughout the study. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, most of the proteins were found to be located in cytoplasm or chloroplast.
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Street, Jonathan Mark. "Exosomal proteome as a source of biomarkers for human disease." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5895.
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Exosomes are small lipid membrane bound vesicles formed as part of the endosomal pathway and released into the extracellular space following fusion of late endosomes with the plasma membrane. Exosomes have been shown to have a variety of biological roles and may represent a novel source of disease biomarkers. The objectives of this project were to develop a panel of techniques for identifying exosomes in human urine then establish an in vitro model to determine whether exosomes change with cellular activation. We then used the techniques developed with human urine to determine whether human cerebrospinal fluid (CSF) contains exosomes and applied a mass spectrometry based approach to characterise the exosomal proteome. We used western blot for three exosomal markers (tsg101, CD24 and flotillin- 1), isopycnic centrifugation on a sucrose density gradient and direct visualisation using transmission electron microscopy (TEM) to verify the presence of exosomes. Using GeLC-MS/MS, 88 proteins were identified in the urinary exosomes. Several of these proteins could be linked to diseases and specific sections of the nephron. A murine cortical collecting duct cell line was used to model exosome release into the urine. Firstly, exosome release was verified using the approach developed in the urine. Stimulation of the cells with desmopressin caused an increase in the presence of aquaporin 2 in the exosomes. This increase reflected a similar change in the cells and occurred over a similar time course. This supports the hypothesis that the exosomes reflect the state of the kidney cells. In contrast, stimulation with cisplatin did not alter the presence of Fetuin-A, a proposed biomarker of cisplatin-induced acute kidney injury, in exosomes and this was consistent with no change in Fetuin- A expression in the cells. The released exosomes may act as mediators of communication to other cells. Following incubation of mCCD cells with AQP2 containing exosomes AQP2 in the cell lysate was increased indicating interaction between the cells and exosomes and potentially internalisation. Exosomes have been shown to be released by neuronal cells in vitro. We identified exosomes in the CSF of humans using western blot for known exosomal markers, density determination and direct visualisation with TEM and Immuno-TEM using an antibody specific for the exosomal marker flotillin-1. Label-free quantitative mass spectrometry was used to compare multiple CSF samples. On a whole protein analysis 86% of the proteins identified varied by less than 2-fold in comparison to the average across samples. On a tryptic peptide analysis 75% of the peptides identified varied by less than 2-fold in comparison with the average across samples. We have demonstrated exosomes are present in urine, CSF and mCCD cell conditioned media. In the mCCD cell derived exosomes we have demonstrated that following stimulation the proteome of the exosomes changes and that this change reflects the change seen in the cells. For the urinary and CSF exosomes we have characterised their proteomes using GeLC-MS/MS. These findings are consistent with the hypothesis that exosomes are a rich source of information, including biomarkers, on their cells of origin.
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Mackenzie, Rebecca. "Investigating the heat shock response of the yeast proteome via quantitative proteomics." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-heat-shock-response-of-the-yeast-proteome-via-quantitative-proteomics(ea5b80a1-6a4a-4207-847e-aefd3e6e4832).html.
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Proteostasis, the regulation of protein abundance and function in cellular systems, underpins the ability of organisms to deal with environmental challenge such as heat shock stress. In this thesis, a quantitative study of this process at the molecular level has been undertaken using the model eukaryote S. cerevisiae to characterise the protein level response to heat shock. Although well studied, much of the previous work has focussed on changes at the mRNA levels or relative changes in protein expression. To address this, an absolute quantification strategy was developed utilising the QconCAT approach. A total of 10 recombinant QconCAT proteins were designed to target the 63 chaperones in S. cerevisiae, with up to 5 Q-peptides selected per chaperone where possible. Subsequently, absolute copy per cell values were determined for 49 of the 63 chaperones in S. cerevisiae under conditions of normal growth and heat shock (42 °C, 30 minutes). Chaperones that are known targets of the heat shock response activating transcription factor HSF1 are significantly upregulated in response to heat shock. Furthermore, this dataset has been extended towards proteome-wide quantification, for which SRM-normalised label free quantification values for 1644 proteins in both conditions were determined. Using these values and a high quality chaperone-client interaction dataset, progress has been made towards modelling the change in the protein volume and workload of each chaperone in response to heat shock. Interestingly, for the chaperone Ssb2, both its workload and absolute abundance were significantly upregulated in response to heat shock. However, across all chaperones, the relationship between protein volume, workload fold change and abundance fold change is minimal; further work is required to investigate this.
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Star, Alexandra. "Enrichment and Identification of Methylation at the Proteome Level." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34178.
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Methylation is a post-translational modification which occurs on lysine and arginine residues. Methylation is difficult to detect due to its low abundance and lack of charge. Our laboratory previously developed a novel enrichment approach, ProMENADe, for lysine and arginine methylation in the human embryonic kidney (HEK) 293T cell line which is coupled with mass spectrometry.
Simplifying a lysate with subcellular fractionation prior to enrichment increased the identification of methylation sites by 39.5% while using multiple proteases for digestion increased identification by 27%. Combining these methods yielded a 47.2% increase. Analysis at the 1% methylation level FDR filtered for C-terminal methylation identified 169 sites and further analysis revealed 74 of these sites overlap with the PhosphoSite database. This ProMENADe enrichment strategy yielded 95 novel methylation sites to the field and can be a key tool in the field of methylation allowing for the enrichment and identification of methylated proteins.
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Sainz-Fuertes, Ricardo. "Applied proteomics : using the peripheral proteome to identify a surrogate marker of schizophrenia." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/applied-proteomics(0e1764b2-7d60-4140-9213-0ac8bc2afd13).html.
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Classical proteomic techniques have been used in medicine for biomarker discovery and have recently entered the arena of neurodegenerative disorders. Biomarkers for disorders such as Huntington’s, Parkinson’s and Alzheimer’s diseases are currently being developed and tested for use in early detection, disease progression, prognosis and response to treatment. However, psychiatric disorders have been less researched to date. In this thesis, a classic proteomic approach was used (1) to examine alterations in molecular pathways determined by a well known high-risk schizophrenia (SCZ) gene (DISCI); (2) to assert the effects of antipsychotic medication in the brain and plasma of F344 rats; and (3) to canvass the plasma of psychotic patients searching for biomarkers of the disease. It was found that DISCI modulated the expression of dihydropteridine reductase, a key enzyme for biogenic amine synthesis and that of peptidyl-prolyl isomerase A, a protein involved in apoptosis. Antipsychotic treatment in rats exerted an effect on glucose and lipid metabolic pathways, mitochondria! function, immune system response, neuronal migration, differentiation and apoptosis. Alterations in calcium signalling pathways were detected in the plasma of psychotic patients, indicated by a significant reduction in plasma levels ofgelsolin and an increment of S100B.
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Koutroukides, Theodoros Alexis. "Serum proteome profiling using amine-reactive isobaric tagging mass spectrometry in schizophrenia." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607699.
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Sherman, James. "Proteome-scale kinetic processes : methods and applications /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/10284.
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Scuderi, Richard Anthony. "The Effects of Diet on the Bovine Milk Proteome." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/846.
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Protein is an important fraction within bovine milk. This milk protein is not only vital for calf growth and development, but also includes bioactive proteins and peptides that have been shown to enhance the health of animals and humans. Research efforts are focusing on factors, such as nutrition, that can influence the quantity and profile of proteins within the bovine milk proteome. The research outlined herein investigated the impact of diet on the bovine milk proteome. The first experiment examined whether dietary inclusion of grape marc (GM), a condensed tannin (CT) containing by-product from the viticulture industry, could alter the bovine milk proteome through altered nitrogen (N) metabolism. In this experiment, 10 lactating Holstein cows were fed either 2.0 kg dry matter (DM)/ cow/ day of beet pulp: soy hulls in a 50% mixture (control), or 1.5 kg DM/ cow/ day of GM as part of a balanced dairy cow ration for a 28-d trial. Milk samples were obtained for analysis of the high- and low-abundance protein fractions. Skimmed milk samples collected for high-abundance protein analysis were measured using high performance liquid chromatography (HPLC), and liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used to identify proteins in the low-abundance protein enriched fraction. Skimmed milk samples collected for low-abundance milk protein analysis were fractionated and enriched to remove higher abundance proteins. Enriched milk samples were then digested and labeled with isobaric tandem mass tags (TMT) prior to protein identification using LC-MS/MS analysis. There were no changes in the high-abundance protein fraction in response to diet; however, 16 of 127 low-abundance proteins were identified at different relative-abundances due to diet (P ≤ 0.05). While there were no alterations in the metabolic or N status of animals due to GM supplementation, the 12% change in the low-abundance milk protein fraction highlighted the potential for dietary alteration of the bovine milk proteome.
A second experiment evaluated the inclusion of alternative forage crops (AFC) as a means to alter the bovine milk proteome. In this experiment, both the skimmed milk and milk fat globule membrane (MFGM) protein fractions were included in analysis. Milk samples were collected from 16 lactating Jersey cattle included in a 21-d grazing experiment, where cows were offered one of two diets. The control group (CON, n=8) grazed a grass-legume pasture mixture containing orchardgrass (Dactylis glomerata), timothy (Phleum pratense), Kentucky bluegrass (Poa pratensis), and white clover (Trifolium repens). The treatment group (AFC, n=8) grazed a similar base pasture that was strip-tilled with oat (Avena sativa), buckwheat (Fagopyrum esculentum), and chickling vetch (Lathyrus sativus) so that the AFC species comprised 10% of the AFC group’s pasture DM intake (DMI). Milk samples were collected for HPLC analysis of the high abundance milk proteins, and LC-MS/MS analysis of the low abundance protein enriched skim milk fraction and MFGM-associated protein fraction. Cows that grazed pastures containing AFC had higher αs1-CAS content (P = 0.005), and higher relative-abundances of 7 low-abundance proteins within the skim milk and MFGM fractions (P ≤ 0.05). While it is plausible that the inclusion of AFC in pasture increased nutrient availability to the mammary gland, the specific mechanisms that could have caused the shifts observed remain unclear. Further investigation is necessary to fully understand the role of diet and the milk protein profile.
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Huang, Peiwu. "Method development and application for spatial proteome and glycoproteome profiling." HKBU Institutional Repository, 2020. https://repository.hkbu.edu.hk/etd_oa/788.
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Tissues are heterogeneous ecosystems comprised of various cell types. For example, in tumor tissues, malignant cancer cells are surround by various non-malignant stromal cells. Proteins, especially N-linked glycoproteins, are key players in tumor microenvironment and respond to many extracellular stimuli for involving and regulating intercellular signaling. Understanding the human proteome and glycoproteome in heterogeneous tissues with spatial resolution are meaningful for exploring intercellular signaling networks and discovering protein biomarkers for various diseases, such as cancer. In this study, we aimed to develop new liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analytical methods for spatially-resolved proteome and glycoproteome profiling in tissue samples, and apply them for profiling potential biomarkers for pancreatic cancer. We first systematically and synchronously optimized the LC-MS parameters to increase peptide sequencing efficiency in data dependent proteomics. Taking advantage of its hybrid instrument design with various mass analyzer and fragmentation strageties, the Orbitrap Fusion mass spectrometer was used for systematically comparing the popular high-high approach by using orbitrap for both MS1 and MS2 scans and high-low approach by using orbitrap for MS1 scan and ion trap for MS2 scans. High-high approach outperformed high-low approach in terms of better saturation of the scan cycle and higher MS2 identification rate. We then systematically optimized various MS parameters for high-high approach. We investigated the influence of isolation window and injection time on scan speed and MS2 identification rate. We then explored how to properly set dynamic exclusion time according to the chromatography peak width. Furthermore, we found that the orbitrap analyzer, rather than the analytical column, was easily saturated with higher peptide loading amount, thus limited the dynamic range of MS1-based quantification. Finally, by using the optimized LC-MS parameters, more than 9000 proteins and 110,000 unique peptides were identified by using 10 hours of effective LC gradient time. The study therefore illustrated the importance of synchronizing LC-MS precursor targeting and high-resolution fragment detection for high-efficient data dependent proteomics. Understanding the tumor heterogeneity through spatially resolved proteome profiling is meaningful for biomedical research. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was stained by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance with traditional H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT was achieved by combining with data independent proteomic analysis. This IHC-SISPROT workflow was successfully applied for identifying 6660 and 6052 protein groups from cancer cells and cancer- associated fibroblasts (CAFs) by using only 5 mm 2 and 12 μm thickness of hepatocellular carcinoma tissue section. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues. N-linked glycoproteins are promising candidates for diagnostic and prognostic biomarkers and therapeutic targets. They often locate at plasma membrane and extracellular space with distinct cell type distribution in tissue microenvironment. Due to access to only low microgram of proteins and low abundance of glycoproteins in tissue sections harvested by LCM, region- and cell type-resolved glycoproteome analysis of tissue sections remains challenging. Here we designed a fully integrated spintip-based glycoproteomic approach (FISGlyco) which achieved all the steps for glycoprotein enrichment, digestion, deglycosylation and desalting in a single spintip device. Sample loss is significantly reduced and the total processing time is reduced to 4 hours, while detection sensitivity and label-free quantification precision is greatly improved. 607 N-glycosylation sites were successfully identified and quantified from only 5 μg of mouse brain proteins. By seamlessly combining with LCM, the first region-resolved N-glycoproteome profiling of four mouse brain regions, including isocortex, hippocampus, thalamus, and hypothalamus, was achieved, with 1,875, 1,794, 1,801, and 1,417 N-glycosites identified, respectively. Our approach could be a generic approach for region and even cell type specific glycoproteome analysis of tissue sections. Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with five year survival rate of around 8%. No effective biomarkers and targeted therapy are one of the major reasons for this urgent clinical situation. To explore potential protein biomarkers and drug targets located at intercellular space of pancreatic tumor microenvironment, we established chemical proteomic approach for deep glycoproteome profiling of PDAC clinical tissue samples based on the above- mentioned new proteomic methods. Taking advantage of a long chain biotin- hydrazide probe with less space hindrance, the new method outperformed traditional hydrazide chemistry method in terms of sensitivity, time efficiency and glycoproteome coverage. The method was successfully applied to enrich and validate LIF and its receptors as potential biomarkers for PDAC. In addition, to explore the full map of pancreatic tumor microenvironment glycoproteome with diagnostic and therapeutic values, we collected 114 pancreatic tissues, including 30 PDAC tumor tissues, 30 adjacent non-tumor (NT) tissues, 32 chronic pancreatitis tissues and 22 normal pancreatic tissues, and systematically profiled their glycoprotein expression pattern by using the developed glycoproteomic strategy. The deepest glycoproteome of PDAC was achieved, which covered the majority of previously reported glycoprotein biomarkers and drug targets for PDAC. Importantly, we discovered many new glycoproteins with differential expression in PDAC and normal tissue types. Moreover, LCM-based cell-type proteome profiling was achieved for 13 PDAC tissue samples, which covered more than 8000 proteins for both pancreatic stromal cells and pancreatic cancer cells in each sample. We therefore provided a valuable resource for screening novel and cancer specific glycoprotein biomarkers for pancreatic cancer with spatial resolution
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So, King-yan Leo, and 蘇敬仁. "Dynamics of the mammalian nuclear proteome during influenza viral infection using SILAC-based MS quantitative proteomics." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47869860.
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Influenza has resided with the human race long before we have any written record of it. Its death toll is one of the highest among all other virus. In recent history, pandemic outbreaks of influenza have caused even more deaths. Therefore it is of great importance that we focus our resources on understanding its viral components and functions.
In this study, chimeric mutagenesis was used to investigate the antigenic variance of antibodies I50C and I131B on H1N1 and H5N1 NP. It was revealed from previous study that antibodies I50C and I131B can detect H1N1 NP but not H5N1 NP. NP from influenza A strains A/Puerto Rico/8/1934 (PR8), A/Vietnam/3046/2004 (3046) and A/Indonesia/5/2005 (indo) were used to construct the NP chimeric mutants. Nucleotide sequence from the region spanning from bp 484-506 was chosen as template to design the primers for obtaining head and tail fragments which were components of the NP constructs. Results showed that antibodies I50C and I131B can only detect NP constructs with PR8 head fragments regardless of any tail fragments, and cannot detect NP constructs with 3046 or indo head fragments. Therefore the binding epitope on H1N1 NP tested by the antibodies I50C and I131B is deduced to be within bp 1-506.
In order to understand the dynamics of host and viral nuclear proteome during the influenza A infection, the pulse SILAC (Stable Isotope Labeling of Amino acids on Cell lines) MS-proteomic approach was adopted. More and more research studies are MS-proteomic based as people recognize that proteins truly define the outcome of a cell, with fewer limitations by solely looking at the genome. The pulse SILAC technique involves incorporating “light” isotope-labeled amino acids such as arginine and lysine into cells’ proteins prior infection experiment. While the cells are under influenza infection, “heavy” isotope-labeled amino acids were used to label the cells 2 hour prior each harvesting time points. Since only proteins synthesized within the 2 hour windows are “heavy” isotope labeled, relative quantification of “heavy” isotope to “light” isotope by mass spectrometry (MS) can be calculated into heavy:light (H/L) ratios. Through this method we can know to what extents are the proteins affected and whether the effect is global or specific. Together with the temporal degree of the data, we can reveal the dynamics of host and viral nuclear proteome during the influenza A infection.
MS results of the influenza viral proteins agree with the viral gene expression profile upon infections and corresponded well with time of viral protein expressions during influenza pathogenesis investigated by other research groups. A number of proteins were identified to increase in turnover rate at 8 hpi. This gives a partial view of up-regulated functions inside the nucleus during influenza A infection at that stage. The up-regulated proteins represent cellular functions that are related to: energy homeostasis, microtubule-dependent transport, DNA coiling regulation, transcription regulation, translation regulation and protein folding.
The findings of this research present more information to understand influenza virus and provide a stepping stone for fellow influenza researchers.published_or_final_version
Pathology
Master
Master of Philosophy
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Grady, Joshua Terrence Wilson. "Adapting Quantitative Protein and Phosphorylation Analyses to a Proteome-Wide Scale." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10852.
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Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) has become the preferred method for large-scale peptide and phosphopeptide identification and quantification. The dominance of LC-MS/MS is the result of improved chromatographic, mass spectrometry and bioinformatic technologies. The applications of these technological improvements drive biological innovation by expanding the realm of possible experimentation, facilitating the creation and evaluation of novel hypotheses. Such improvements are the focus of this dissertation. New technologies are presented and their proteome wide applications in biological systems are demonstrated. A comparison of common phosphopeptide enrichment methods is presented in chapter two, which demonstrates that a combination of methods provides non-overlapping data sets. This comparison was performed in mitotically arrested fission yeast, a previously unstudied system by phosphoproteomic methods. This chapter remarks upon phosphorylation site conservation between lower and higher eukaryotes, as a means of predicting potentially relevant phosphorylation events in mammals. A new protocol for tissue based peptide quantification is presented in chapter three. The large-scale application of this method is detailed in a system of mouse liver phosphorylation, between fasted and re-fed states. The effect of peptide and protein level false discovery rates on the accuracy of phosphorylation site quantification is highlighted. This method is a cost-effective alternative to available techniques, such as metabolic labeling, and expands the application of proteomics to include larger animals. Finally, an in depth analysis of quantitative LC-MS/MS based multiplexing is the subject of the last chapter. New techniques for peptide pre-fractionation and ion quantification are discussed, which improve proteome coverage and quantitative accuracy. This proteome-wide multiplexing is applied to an analysis of the budding yeast environmental stress response. Applicable methods of data processing and a means of obtaining biologically relevant information out of multidimensional proteomic data sets are discussed. In all chapters, the data presented represent the largest analyses of their kind. This dissertation provides a solid guide for future proteome-wide studies, focused on the identification and quantification of peptides and their posttranslational modifications.
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Mujahid, Hana, Feng Tan, Jian Zhang, Babi Ramesh Nallamilli, Ken Pendarvis, and Zhaohua Peng. "Nuclear proteome response to cell wall removal in rice (Oryza sativa)." BioMed Central, 2013. http://hdl.handle.net/10150/610238.
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Plant cells are routinely exposed to various pathogens and environmental stresses that cause cell wall perturbations. Little is known of the mechanisms that plant cells use to sense these disturbances and transduce corresponding signals to regulate cellular responses to maintain cell wall integrity. Previous studies in rice have shown that removal of the cell wall leads to substantial chromatin reorganization and histone modification changes concomitant with cell wall re-synthesis. But the genes and proteins that regulate these cellular responses are still largely unknown. Here we present an examination of the nuclear proteome differential expression in response to removal of the cell wall in rice suspension cells using multiple nuclear proteome extraction methods. A total of 382 nuclear proteins were identified with two or more peptides, including 26 transcription factors. Upon removal of the cell wall, 142 nuclear proteins were up regulated and 112 were down regulated. The differentially expressed proteins included transcription factors, histones, histone domain containing proteins, and histone modification enzymes. Gene ontology analysis of the differentially expressed proteins indicates that chromatin & nucleosome assembly, protein-DNA complex assembly, and DNA packaging are tightly associated with cell wall removal. Our results indicate that removal of the cell wall imposes a tremendous challenge to the cells. Consequently, plant cells respond to the removal of the cell wall in the nucleus at every level of the regulatory hierarchy.
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Honan, Mallory Cate. "Examination Of Bovine Rumen Fluid And Milk Fat Globule Membrane Proteome Dynamics." ScholarWorks @ UVM, 2019. https://scholarworks.uvm.edu/graddis/1164.
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Proteomic technology has been increasingly incorporated into agricultural research, as characterization of proteomes can provide valuable information for potential biomarkers of health and physiological status of an animal. As dairy cattle are a dominant production animal in the USA, their biofluids such as milk, blood, urine, and rumen fluid have been examined by proteomic analysis. The research outlined herein was performed to further characterize the dynamics of specific proteomes and relate them to dairy cattle physiology.
The first experiment evaluated the diurnal dynamicity of the rumen metaproteome in Holstein dairy cattle. Rumen fluid was collected from three mid to late lactation multiparous dairy cattle (207 ± 53.5 days in milk) at three time points relative to their first morning offering of a total mixed ration (TMR) (0 h, 4 h, and 6 h after feeding). Samples were processed and labeled using Tandem Mass tagging before being further fractionated with a high pH reversed-phase peptide fractionation kit. Samples were analyzed by LC-MS/MS and statistically analyzed for variations across hour of sampling using the MIXED procedure of SAS with orthogonal contrasts. A total of 242 proteins were characterized across 12 microbial species, with 35 proteins identified from a variety of 9 species affected by time of collection. Translation-related proteins were correlated positively with increasing hour of sampling while more specific metabolic proteins were negatively correlated with increasing hour of sampling. Results suggest that as nutrients become more readily available, microbes shift from conversion-focused biosynthetic routes to more encompassing DNA-driven pathways.
The second experiment aimed to characterize the milk fat globule membrane (MFGM) proteomes of colostrum and transition milk for comparison from multi- (n = 10) and primiparous (n = 10) Holstein dairy cattle. Samples were collected at four timepoints post-partum (milkings 1, 2, 4, and 14). After isolation of the protein lysates from the MFGM, proteins were labeled using Tandem Mass tagging and analyzed using LC-MS/MS techniques. Protein identification was completed using MASCOT and Sequest in Proteome Discoverer 2.2. Protein abundance values were scaled and analyzed using the MIXED procedure in SAS to determine the effect of parity, milking number, and parity x milking number, and the adaptive false-discovery rate (FDR)-adjusted P values were determined using the MULTTEST procedure of SAS. There were 104 proteins identified within the MFGM. Statistical analysis revealed that 44.2% of proteins were affected by parity, 70.2% by milking number, and 32.7% by the variable of parity x milking number. There was a two-fold difference in calcium sensing S100 proteins in cows differing in parity possibly due to the multiparous mammary gland being more adapted to the physiological demand of lactation or the lesser requirement of calcium in primiparous cows because of a lower production rate.
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Schlüsener, Daniela. "Proteomics of membrane proteins: development and application for the membrane proteome of Corynebacterium glutamicum." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=978723678.
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Berglund, Lisa. "Selection of antigens for antibody-based proteomics." Doctoral thesis, Stockholm : School of Biotechnology, Royal Institute of Technology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4706.
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Sun, Benjamin Boyang. "Genetic determinants of the human plasma proteome and their application in biology and disease." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/269287.
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Proteins are the primary functional units of biology and the direct targets of most drugs, yet there is limited knowledge of the genetic factors determining inter-individual variation in protein levels (protein quantitative trait loci (pQTLs)). Limitations in high-throughput proteomic measurement technology have meant well-powered genome-wide association studies for large number of proteins so far have lagged behind many of the other "omic" studies such as transcriptomics and metabolomics. This is made more challenging by the complexity of human plasma, characterised by high dynamic range spanning several magnitudes of concentrations and a large number of low abundance proteins. By using an expanded high-throughput multiplex aptamer-based proteomic assay with more than twice the proteome coverage of previous studies, I am able to greatly expand on existing knowledge on genetic determinants of human plasma proteins through testing 10.6 million DNA variants against levels of 2,994 proteins in 3,301 individuals. I identify 1,927 genetic associations with 1,478 proteins, replicating many previous associations as well as gaining novel insights into the genetic architecture of the human plasma proteome. I use several approaches to highlight the application of pQTLs to biology and disease. I show several examples linking distant pQTLs to biologically plausible genes and demonstrate the mediation of distant pQTL by local protein levels, highlighting the role of protein-protein interactions. In addition, I find epistatic effects of genetically determined phenotypes (blood group and secretor status) on protein levels. Through linking previous disease associations, I show that disease associated variants are enriched for pQTLs and I provide insights into possible mechanisms underpinning some of the disease loci. Finally, I identify causal roles for protein biomarkers in disease through multivariable Mendelian randomisation (MR) analysis, leveraging on the simultaneous measurement of multiple functionally related proteins in a locus to account for potential pleiotropic effects. Whereas MR studies of plasma proteins have been constrained by availability of few suitable genetic instruments, the data generated here remedy this bottleneck by furnishing an extensive toolkit. Overall, the work within this thesis foreshadows major advances in post-genomic science through increasing application of novel bioassay technologies to major population biobanks.
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Leary, Dagmar Hajkova. "CIRCADIAN PROTEOME CHANGES IN PHOTORECEPTOR OUTER SEGMENTS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1264276011.
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Lai, Kwok-wai, and 賴國偉. "Proteome analysis of anther-development-related proteins in a thermo-sensitive male sterile rice mutant." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B29517862.
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Tuli, Leepika. "Proteome Profiling of Saccharomyces cerevisae stress response to Cumene Hydroperoxide (CHP)." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/28620.
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Oxidative stress, described as the state of disturbed intracellular redox balance, has been associated with several human conditions including ageing, apoptosis, cancer, autoimmune and neuro-degenerative diseases. Stress studies have shown that reactive oxygen species (ROS) and reactive nitrogen species (RNS) along with its intermediates can attack essential cell targets such as: DNA, proteins, lipids and carbohydrates, leaving behind dysfunctional biologic molecules. In effect, a cellâ s primary response is to involve several defense mechanisms that are under a complex and intricate regulatory control to repair any damages that may have occurred. Although several stress studies have been conducted in the past that have approached this biologically complex process step by step, application of a Systems Biology towards a comprehensive understanding is still emerging.
The current objective of this project is to identify proteins that change in response to cumene hydroperxoide (CHP) treatment and in parallel make an attempt to uncover events and processes that are a part of CHP-induced oxidative stress response. From a systems biology viewpoint, the Yeast Oxidative Stress project will monitor response at three different levels: transcriptomics, proteomics and metabolomics, with dynamic changes being measured from 3 to 120 min after CHP addition. Data collected from the different levels will be integrated to accomplish a holistic viewpoint of stress response in the given system and to develop mathematical tools for modeling biochemical networks.
Saccharomyces cerevisiae was chosen as a model, based on its availability of a completely mapped genome sequence with a collection of null mutants that was relevant to our fundamental research of stress response mechanism. Yeast, a simple unicellular eukaryote has been extensively used for applied studies and has proven to be indispensable for stress research. Information derived from this project can reveal response mechanisms used by higher eukaryotes, especially if via analogous signaling cascades that are comparable between organisms.
Current research investigates an optimal workflow for generating 2D gel based protein expression data and identifying proteins that are induced by cumene hydroperoxide treatment. A non-targeted protein profiling followed by a 2-way ANOVA analysis provided a list of proteins that differ significantly between treatments. Protein identification provided relevant information on which proteins are affected by CHP induced stress response, including posttranslational modifications of peroxiredoxins. Redox active protein, Ahp1, was regulated post-translationally with sulfonic acid modification observed for its active Cys(62) residue.Ph. D.
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Sarvaiya, Hetal Abhijeet. "Mass Spectrometric Characterization of the MCF7 Cancer Cell Line: Proteome Profile and Cancer Biomarkers." Thesis, Virginia Tech, 2006. http://hdl.handle.net/10919/42169.
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The discovery of cancer biomarkers is crucial in the clinical setting to facilitate early diagnosis and treatment, thereby increasing survival rates. Proteomic technologies with mass spectrometry detection (MS) have the potential to affect the entire spectrum of cancer research by identifying these biomarkers. Simultaneously, microfabricated devices have evolved into ideal analysis platforms for minute amounts of sample, with promising applications for proteomic investigations and future biomarker screening. This thesis reports on the analysis of the proteomic constituents of the MCF7 breast cancer cell line using a shotgun 2-D strong cationic exchange/reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (SCX/RP-LC-ESI-MS/MS) protocol. A series of optimization strategies were performed to improve the LC-MS experimental set-up, sample preparation, data acquisition and database searching parameters, and to enable the detection and confident identification of a large number of proteins. Over ~4,500 proteins were identified using conventional filtering parameters, and >2000 proteins using a combination of filters and p-value sorting. Of these, ~1,950 proteins had p<0.001 (~90%) and more than half were identified by ≥ 2 unique peptides. About 220 proteins were functionally involved in cancer related cellular processes, and over 100 proteins were previously described in the literature as potential cancer markers. Biomarkers such as PCNA, cathepsin D, E-cadherin, 14-3-3-sigma, antigen Ki-67, TP53RK, and calreticulin were identified. These data were generated by subjecting to mass spectrometric analysis ~42 µg of protein digest, analyzing 16 SCX peptide fractions, and interpreting ~55,000 MS2 spectra. Total MS time required for analysis was 40 h.
Selective SCX fractions were also analyzed by using a microfluidic LC platform. The performance of the microchip LC was comparable to that obtained with bench-top instrumentation when similar experimental conditions were used. The identification of 5 cancer biomarkers was enabled by using the microchip LC platform. Furthermore, this device was also capable to analyze phosphopeptides.Master of Science
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Modak, Swananda Rajan. "Effects of Aβ42 on the human proteome and compound library screening using cellular models of Alzheimer's disease." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/effects-of-a42-on-the-human-proteome-and-compound-library-screening-using-cellular-models-of-alzheimers-disease(4cca38b1-fd3f-4ef1-bde1-e16cf227ef68).html.
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The neuropathological process in Alzheimer's disease (AD) is characterized by both intra and extracellular Aβ42 aggregates. The neuropathological process of AD is complex and the exact cause of Aβ aggregation leading towards neuronal death is yet unknown. Several events are implicated towards the development of AD including changes within the proteome. With more than 30 million people currently affected with AD, there is still no cure for AD. In this project we seek to identify differential protein profiles by undertaking a comparative analysis of the intracellular and extracellular effects of Aβ on the human proteome using two cellular neuronal models: MC65 and SHSY5Y cells, to understand the biochemical pathology underlying AD. We also initiated a compound screening approach which not only identified several small molecules and peptides inhibiting the Aβ cytotoxicity, but also identified several known compounds from the LOPAC library acting as potential inhibitors of intra and extracellular Aβ42 cytotoxicity, thus highlighting the importance of drug repositioning to identify novel compounds in the therapeutic regime of AD which could be categorized as Aβ toxicity inhibitors. A comparative qualitative proteomics approach was undertaken using OFFGEL fractionation. The MS data was analysed through GO, biological pathway and protein interaction analysis using various databases such as UniProtKB, DAVID v6.7, KEGG and String 9.0 for the SHSY5Y cells treated with extracellular Aβ42 and MC65 cells which conditionally express intracellular C99, that is further cleaved to intracellular Aβ. This was followed by validation of 8 proteins by in-cell Western assay (ICW) undertaken using the LI-COR Infrared Imaging System for the cell lysates of control and Aβ42 treated SH-SY5Y as well as Aβ induced MC65 cells. We have also screened a library of 1280 LOPAC compounds on both the cell lines and 9 other compounds previously known as Aβ toxicity inhibitors on MC65 cells. The lead compounds were further characterized using MTT, LDH, ThT and ICW assays. The proteomics methodology undertaken through this project identified several novel proteins specific to intracellular and extracellular Aβ aggregation. The GO, biological pathway analysis and the functional interaction study helped to identify proteins associated from the proteasome pathway to be affected as an effect of Aβ aggregation for both the cells exposed with intra and extracellular Aβ aggregation. The compound screening study also identified several compounds as inhibitors of Aβ cytotoxicity. A-77636, a D1 dopamine receptor agonist was identified as a lead compound to reduce the extracellular Aβ42 cytotoxicity at nM concentration. Moreover, 1,3-Diethyl-8-phenylxanthine and Arecaidine propargyl ester hydrobromide also proved successful in attenuating the extracellular Aβ42 cytotoxicity. Apart from this; SEN1000, SEN304 and Scylloinositol were able to completely attenuate the intracellular Aβ cytotoxicity, whereas two other compounds, 1,3-Dipropyl-8-p-sulfophenylxanthine and 3-Bromo-7-nitroindazole from the LOPAC library proved effective in acting as partial inhibitors of intracellular Aβ aggregation induced cytotoxicity. The ADME profile for most of these compounds is acceptable, therefore these can be considered as therapeutic leads for AD in the future.
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Macarthur, Deborah Jane. "Mapping the proteome of Streptococcus gordonii." University of Sydney. Health Science, 2005. http://hdl.handle.net/2123/686.
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Streptococcus gordonii is a primary coloniser of the tooth surface where it efficiently ferments carbohydrates at pH levels above 6.0. By not being able to maintain the pH of dental plaque to a level required for enamel dissolution, the dominance of S. gordonii in dental plaque is considered a sign of a healthy oral cavity. However, upon entering the bloodstream and encountering a rise in pH, S. gordonii may become pathogenic, being one of the major causative organisms associated with infective endocarditis. Proteome analyses of S. gordonii grown at steady state in a chemostat allowed the phenotypic changes associated with alterations in pH levels characteristic of these two environments to be determined. As an initial starting point to this study, a two-dimensional electrophoresis (2- DE) reference map of S. gordonii grown at pH 7.0 was produced. Although only 50% of the S gordonii genome was available in an annotated form during the course of this study, the closely related Streptococcus pneumoniae genome (with which S. gordonii shares 97.24% DNA sequence homology) had been completed in 2001. The use of both of these databases allowed many of the S. gordonii proteins to be identified by mass spectrometry. Four hundred and seventy six protein spots, corresponding to 250 different proteins, or 12.5% of the S. gordonii proteome, were identified, giving rise to the first comprehensive proteome reference map of this oral bacterium. Of the 250 different proteins, 196 were of cellular origin while 68 were identified from the extracellular milieu. Only 14 proteins were common to both compartments. Of particular interest among the 54 uniquely identified extracellular proteins was a homologue of a peptidoglycan hydrolase that has been associated with virulence in S. pneumoniae. Among the other proteins identified were ones involved in transport and binding, energy metabolism, translation, transformation, stress response and virulence. Twelve cell envelope proteins were identified as well as 25 others that were predicted to have a membrane association based on the presence of at least one transmembrane domain. The study also confirmed the existence of 38 proteins previously designated as �hypothetical� or with no known function. Mass spectral data for over 1000 protein spots were accumulated and archived for future analysis when sequencing of the S. gordonii genome is finally completed. Following the mapping of the proteome of S. gordonii, alterations in protein spots associated with growth of the bacterium at pH intervals of 0.5 units in the pH range 5.5 - 7.5 were determined. Only 16 protein spots were shown to be significantly altered in their level of expression despite the range of pH studied. Among the differentially expressed proteins was a manganese-dependent inorganic pyrophosphatase (PpaC), which regulates expression of adhesins required for coaggregation. The expression of PpaC was highest at pH 6.5 - 7.0, the pH of a healthy oral cavity, indicating that PpaC may play an important part in dental plaque formation. Another differentially expressed protein was the heat-inducible transcription repressor (HrcA). Alterations in HrcA were consistent with its role as a negative repressor in regulating heat-shock proteins at low pH, even though no changes in the level of heat-shock proteins were observed as the pH declined. This result gave rise to the hypothesis that the possible reason cariogenic bacteria, such as Streptococcus mutans, can out compete S. gordonii at low pH might simply be due to their ability to manipulate their proteome in a complex manner for survival and persistence at low pH, unlike S. gordonii. This may imply some prevailing level of genetic regulation that is missing in S. gordonii.
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Macarthur, Deborah Jane. "Mapping The Proteome Of Streptococcus Gordonii." Thesis, The University of Sydney, 2005. http://hdl.handle.net/2123/5097.
Full textAbstract:
Streptococcus gordonii is a primary coloniser of the tooth surface where it efficiently ferments carbohydrates at pH levels above 6.0. By not being able to maintain the pH of dental plaque to a level required for enamel dissolution, the dominance of S. gordonii in dental plaque is considered a sign of a healthy oral cavity. However, upon entering the bloodstream and encountering a rise in pH, S. gordonii may become pathogenic, being one of the major causative organisms associated with infective endocarditis. Proteome analyses of S. gordonii grown at steady state in a chemostat allowed the phenotypic changes associated with alterations in pH levels characteristic of these two environments to be determined. As an initial starting point to this study, a two-dimensional electrophoresis (2- DE) reference map of S. gordonii grown at pH 7.0 was produced. Although only 50% of the S gordonii genome was available in an annotated form during the course of this study, the closely related Streptococcus pneumoniae genome (with which S. gordonii shares 97.24% DNA sequence homology) had been completed in 2001. The use of both of these databases allowed many of the S. gordonii proteins to be identified by mass spectrometry. Four hundred and seventy six protein spots, corresponding to 250 different proteins, or 12.5% of the S. gordonii proteome, were identified, giving rise to the first comprehensive proteome reference map of this oral bacterium. Of the 250 different proteins, 196 were of cellular origin while 68 were identified from the extracellular milieu. Only 14 proteins were common to both compartments. Of particular interest among the 54 uniquely identified extracellular proteins was a homologue of a peptidoglycan hydrolase that has been associated with virulence in S. pneumoniae. Among the other proteins identified were ones involved in transport and binding, energy metabolism, translation, transformation, stress response and virulence. Twelve cell envelope proteins were identified as well as 25 others that were predicted to have a membrane association based on the presence of at least one transmembrane domain. The study also confirmed the existence of 38 proteins previously designated as �hypothetical� or with no known function. Mass spectral data for over 1000 protein spots were accumulated and archived for future analysis when sequencing of the S. gordonii genome is finally completed. Following the mapping of the proteome of S. gordonii, alterations in protein spots associated with growth of the bacterium at pH intervals of 0.5 units in the pH range 5.5 - 7.5 were determined. Only 16 protein spots were shown to be significantly altered in their level of expression despite the range of pH studied. Among the differentially expressed proteins was a manganese-dependent inorganic pyrophosphatase (PpaC), which regulates expression of adhesins required for coaggregation. The expression of PpaC was highest at pH 6.5 - 7.0, the pH of a healthy oral cavity, indicating that PpaC may play an important part in dental plaque formation. Another differentially expressed protein was the heat-inducible transcription repressor (HrcA). Alterations in HrcA were consistent with its role as a negative repressor in regulating heat-shock proteins at low pH, even though no changes in the level of heat-shock proteins were observed as the pH declined. This result gave rise to the hypothesis that the possible reason cariogenic bacteria, such as Streptococcus mutans, can out compete S. gordonii at low pH might simply be due to their ability to manipulate their proteome in a complex manner for survival and persistence at low pH, unlike S. gordonii. This may imply some prevailing level of genetic regulation that is missing in S. gordonii.
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Chitteti, Brahmananda Reddy. "Proteome and phosphoproteome dynamic change during cell dedifferentiation in Arabidopsis thaliana." Diss., Mississippi State : Mississippi State University, 2007. http://library.msstate.edu/etd/show.asp?etd=etd-07172007-035002.
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Wood, Laura Charlotte. "Understanding kinetochore dependency pathways using vertebrate conditional knockout cell lines and quantitative proteomics." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/8964.
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When cells divide, a series of events must proceed in a timely and co-ordinated manner to ensure that all DNA is replicated and partitioned equally between the two daughter cells. A central component of this process is the kinetochore, a large proteinaceous complex (>100 proteins) found within the centromere of all chromosomes. During the dynamic process of cell division, this machinery must be able to capture microtubules, promote chromosome movements towards the spindle midzone and ensure that segregration only occurs once this alignment has been successfully completed. This requires intricate mechanical and regulatory co-ordination between components and it is therefore no surprise that the structures responsible are structurally and functionally varied. It has, however, become clear that many kinetochore proteins assemble into distinct sub-complexes and despite the fact that their specific contributions are well studied, the way the many unique sub-assemblies come together to form a fully operational kinetochore is still poorly understood. Here, chromosome isolation techniques from chicken DT40 cells combined with mass spectrometry employing Stable Isotope Labeling by Amino acids in Cell culture (SILAC), is used to compare the proteome of mitotic chromosomes from different conditional kinetochore knockout (KO) cell lines. This includes components of the inner kinetochore; CENP-C, CENP-T and CENP-W, and a sub-unit of the Ndc80 complex that is important for microtubule attachment. With these large data sets I have focused on the impact these depletions have on the architecture of the holo-kinetochore by measuring the SILAC ratios of individual proteins. From these measurements I can define whether specific components are decreased, increased or unchanged in terms of their abundance on chromosomes in response to the various deletions. I have found that proteins within the same complex typically behave in a similar manner across the different KO conditions. By integrating all of the data sets, dependency networks are revealed, as well as highlighting potential novel kinetochore proteins worthy of further study.
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Schneider, Sonja [Verfasser], and Stefan [Akademischer Betreuer] Stevanovich. "Plasma Proteome Analysis Using Peptide Group-Specific Immunoprecipitation "Triple X Proteomics" / Sonja Schneider ; Betreuer: Stefan Stevanovich." Tübingen : Universitätsbibliothek Tübingen, 2012. http://d-nb.info/1162842873/34.
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Agana, Bernice A. "Mass Spectrometry-Based Proteomics and Metabolomics: Understanding Protein Interactions, Proteome Complexity and Perturbations in Cellular Metabolism." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574636665012436.
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Fava, Marika. "Proteomics analysis of intracellular and extracellular proteome in aneurysmal patients with bicuspid and tricuspid aortic valve." Thesis, St George's, University of London, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706521.
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Background. Thoracic aortic aneurysm (TAA) is a degenerative disease of the aortic wall and is associated with an increased risk of aortic rupture. TAA is a more prevalent complication in patients with bicuspid aortic valve (BAV) compared to those with tricuspid aortic valve (TAV). Objectives. We aimed to investigate first, changes in intracellular proteins by comparing BAV and TAV patients with aneurysm, and second, regional changes in extracellular matrix (ECM) proteins by comparing the lesser (concavity) and the greater (convexity) aortic curvatures of BAV patients with and without aneurysm. The findings obtained in human patients were followed up by mechanistic studies in mice. Methods. Human and murine aortic specimens were analysed by mass spectrometry (MS) after extraction of intracellular and ECM proteins. Immunoblotting and gene expression analysis were used to validate the proteomics findings. An ECM protease knockout mouse model was used to better characterise proteolytic processing of ECM large proteoglycan. Similar techniques were used as above. Results. To compare the intracellular proteome of aneurysmal BAV and TAV patients we used two-dimensional fluorescence difference in-gel electrophoresis. This approach revealed regulation of 24 proteins. BAV aneurysms were associated with up-regulation of smooth muscle cell related proteins and downregulation of glycolytic and oxidative stress related proteins. Next, we focused on alterations in ECM proteins and compared the concave and convex areas in aortas of BAV patients with and without TAA using liquid chromatography and tandem MS. In aortas of aneurysmal BAV patients, versican, a large proteoglycan important for tissue integrity, was up-regulated in the concave area; conversely, versican was down-regulated in the same area of non-aneurysmal BAV samples. Gene expression analysis showed no differences in versican levels between concavity and convexity of aneurysmal patients, suggesting that regulation of versican protein levels may be related to proteolytic processing. ADAMTSs (a disintegrin and metalloproteases with thrombospondin motifs) are the main proteases cleaving versican. Notably, in aneurysmal BAV patients there was an inverse association between the abundance of versican and gene expression levels of members of the ADAMTS family. To study the role of ADAMTS in the remodelling of the ECM in aorta, we used ADAMTS-5 knockout mice. After angiotensin II infusion to induce aortic dilatation, versican levels were higher in aortas of ADAMTS-5 knockout mice activity compared to wild-type controls. Conclusion. Our results suggest that ADAMTS proteases may be important for versican regulation during aortic ECM remodelling, as supported by proteomics changes in the ECM of aneurysmal BAV patients and ADAMTS-5 knockout mice.
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Aras, Taskin Asli. "Proteome-wide Analysis Of Functional Roles Of Bacilysin Biosynthesis In Bacillus Subtilis." Master's thesis, METU, 2010. http://etd.lib.metu.edu.tr/upload/12612409/index.pdf.
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The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. Most of these metabolites are small peptides that have unusual components and chemical bonds and synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is one of the simplest peptide antibiotics known. It is a dipeptide with an N-terminal L-alanine and an unusual amino acid, L-anticapsin, at its C-terminal. Recently, ywfBCDEF operon of B. subtilis 168 was shown to carry bacilysin biosynthesis function, the genes of this operon were renamed as bacABCDE. The first member of bac operon, bacA gene was proved to encode the function of L-alanine &ndashL-anticapsin amino acid ligation. Bacilysin production is regulated at different levels, negatively by GTP via the transcriptional regulator CodY and AbrB while positive regulation occurs by guanosine 5
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Pizol, José Vitor. "The effects of rockrose extract feed inclusion on the wool proteome of portuguese White Merinos." Master's thesis, ISA, 2020. http://hdl.handle.net/10400.5/21503.
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Mestrado em Engenharia Zootécnica - Produção Animal / Instituto Superior de Agronomia / Faculdade de Medicina Veterinária. Universidade de LisboaThis study was carried out with the objective of evaluating the effect of feeding extract of rockrose (Cistus ladanifer L.), a perennial shrub commonly found in the marginal regions of Portugal and rich in condensed tannins (CT), and its effect on the wool proteome of Portuguese White Merinos. Twenty-four Portuguese White Merinos lambs were fed a diet restricted in protein and composed of grass hay (15%) and concentrate (85%), where they were distributed in three experimental groups: control group, composed of 16% crude protein (CP); diet restricted to 12% crude protein (RP) and diet restricted to 12% crude protein and treated with 1.5% rockrose extract (RPCT). After the 5-week experimental period, samples of wool present in an area of approximately 100cm² in the anterior region of the head were collected from each animal and used for subsequent analysis of proteomics based on liquid chromatography - mass spectrometry (LC-MS). The proteomic method identified 110 proteins in the wool fibre. Of this total, 4 showed significance (P<0.05) and could be classified as structural proteins, with three Type I keratins (Keratin 36, Keratin Type I and Keratin 10) and one Keratin Associated Protein (KAP9.2). The differential accumulation results showed that the RPCT group decreased the expression of these proteins in relation to the Control and RP groups. Except for the K36 protein, which had a 19% higher expression for the RPCT group compared to the other two groups. We conclude that the rockrose extract has a minor effect on the wool proteome that affect specifically the parts of the wool fibre where these proteins are located with putative changes in fibre structure and properties. Finally, further microscopy and wool quality traits studies are still needed to confirm and explain these changes
N/A
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So, Wing-yan. "Proteome and gene expression analysis in white adipose tissue of diet-induced obese mice." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/hkuto/record/B39367435.
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Öztürk, Özgür. "Feature extraction and similarity-based analysis for proteome and genome databases." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1190138805.
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Romas, Laura. "Understanding the mucosal fluid proteome in rectal susceptibility to HIV infection." PLoS One, 2014. http://hdl.handle.net/1993/30854.
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Objective: The rectal mucosa is highly susceptible to HIV infection. Mucosal fluid contains soluble immune proteins that influence HIV infection, and previous studies have shown unique mucosal protein expression in HIV-exposed seronegative (HESN) populations, which may contribute to reduced HIV susceptibility. However, the key correlates of susceptibility at the rectal mucosa have not been well defined, which is a critical knowledge gap for our understanding of HIV pathogenesis. Methods: Rectal lavage from low risk men was screened for HIV-neutralizing activity in a TZM-bl reporter cell line against an R5-tropic HIV virus. Label-free tandem mass spectrometry was used to characterize soluble proteins within rectal lavage samples from a low-risk cohort of men (n=15), and HESN men who have sex with men (MSM; n=25). Protein expression between populations was compared using adjusted t tests (p<0.05), and was interpreted using hierarchical clustering and DAVID biofunctional analysis. Protein expression was further analyzed using survey data on sexual behaviours. Proteins associated with the HESN population were screened for antiviral activity in TZM-bl and PBMC culture against an R5- and X4-tropic virus. Major Results: Rectal mucosal fluid was able to inhibit HIV infection in vitro by 40% (p<0.05). Mass spectrometry identified 30/341 (9%) proteins deferentially expressed (DE) in HESN MSM. DE proteins held functions in immunity (p=6.68x10-6, p=0.001) and epithelial barrier development (p=1.81x10-4; p=0.01); notably, specific antiproteases were elevated in HESN secretions, two of which were screened for antiviral activity. Serpin B4 (+2.52 L2FD; p=1.09x10-5), showed significant inhibition of HIV in TZM-bl (45% BaL, 34% IIIB; p<0.05) and PBMC culture (37% BaL, 49% IIIB; p<0.05); cystatin A (+1.52 L2FD; p=1.40x10-3) showed no inhibitory effects. Serpin B4 expression was not associated with frequency of oral intercourse (p=0.32), partner viral load (r=0.16; p=0.29) or presence of HIV neutralizing IgA in secretions (p=0.52). Conclusions: This thesis reports the use of proteomics to understand HIV-susceptibility at the rectal mucosa, and identified serpin B4 as a novel antiviral immune correlate in a population of HESN MSM. These results may help guide future studies of prevention technologies, such as microbicides or vaccines, which would ultimately help limit the spread of HIV.February 2016
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Arruda, Paulo Cesar Lopes de. "Effects of different lipid sources on housing characteristics, lipid profile and proteome Longissimus Dorsi lambs woolless." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=16272.
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FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgicoThe study was conducted to evaluate the influence of supplementation of different lipid sources on performance and quantitative characteristics of housing and not housing, fatty acid profile and muscle proteome Longissimus dorsi of Santa Ines lambs fed different additional sources of lipids. 35 lambs bulls were used, with initial body weight (13 Â 1.80 kg) and approximately two months in a randomized block design with five treatments and seven repetitions. The treatments consisted of five diets, one free additional lipids (control) and the other, added cottonseed (CA), bran cashew (FCC), cottonseed with cashew nut meal (CALFCC) and calcium salts of long chain fatty acids (LCFA-Ca). The duration of the experiment was determined by the time required for the average body weight of all animals in each treatment were 28 kg, and this selected group for slaughter. No significant effect was observed with the use of supplementary sources of lipids on the loss of fasting, body weight at slaughter, empty body weight, hot carcass weight, cold carcass weight loss by cooling and biological yield. The non-carcass components, intestines and organs showed no differences across lipid additional sources. Regarding the fatty acid profile were observed 13 fatty acids, four of which are saturated, monounsaturated five-four polyunsaturated (PUFA). In proteome analysis, the proteins were expressed in greater quantity were located primarily at pH less than 5.5. Comparing the expression intensity spots between treatments can observe significant difference in the intensity of the spots 23 for the diet revealed fifteen proteins and of this total, seven were differentiated with respect to time. The proteins identified in this experiment can be mainly classified as structural cellular organization and protection to stress, as well as specific metabolic functions and other functions. The addition of various sources of lipid influenced the productive performance and features: hot carcass dressing and cold, yield and weight of the rack the weight and performance of the gastrointestinal tract filled, small intestine and liver income in Santa Ines sheep growing. Supplementation with different lipid sources influence the profile of fatty acids of Longissimus dorsi, so that diets containing FCC and Lcfa-Ca increased the amount of CLA in the muscle. The proteome analysis may be a method to identify marker proteins meat quality prediction. In this study, the identified proteins are highly relevant to the meat tenderness process, wherein the meat maturation process is complex and involves protein in different biological processes. The type of power had greater influence on the proportions of bodies responsible for digestion and absorption of nutrients.
O estudo foi realizado com objetivo de avaliar a influÃncia da suplementaÃÃo de diferentes fontes de lipÃdeos sobre o desempenho produtivo e as caracterÃsticas quantitativas de carcaÃa e nÃo carcaÃa, perfil de Ãcidos graxos e proteoma do mÃsculo Longissimus dorsi de cordeiros Santa InÃs alimentados com diferentes fontes suplementares de lipÃdeos. Foram utilizados 35 cordeiros nÃo-castrados, com peso corporal mÃdio inicial (13  1,80 kg) e aproximadamente, dois meses de idade em delineamento de blocos ao acaso com cinco tratamentos e sete repetiÃÃes. Os tratamentos experimentais consistiram de cinco raÃÃes, sendo uma isenta de lipÃdeos suplementares (controle) e as demais, adicionadas de caroÃo de algodÃo (CA), farelo da castanha de caju (FCC), caroÃo de algodÃo com farelo de castanha de caju (CALFCC) e sais de cÃlcio de Ãcidos graxos de cadeia longa (Ca-Agcl). A duraÃÃo do experimento foi determinada pelo tempo necessÃrio para que a mÃdia do peso corporal de todos os animais de cada tratamento atingisse 28 kg, sendo este grupo selecionado para o abate. NÃo foi observado efeito significativo com a utilizaÃÃo de fontes suplementares de lipÃdeos sobre a perda do jejum, peso corporal ao abate, peso de corpo vazio, peso de carcaÃa quente, peso de carcaÃa fria, perda por resfriamento e rendimento biolÃgico. Os componentes nÃo-carcaÃa, vÃsceras e ÃrgÃos, nÃo apresentaram diferenÃas em funÃÃo das fontes suplementares lipÃdicas. No tocante ao perfil de Ãcidos graxos foram observados 13 Ãcidos graxos, dos quais quatro sÃo saturados, cinco monoinsaturados e quatro poliinsaturados(AGPI). Na anÃlise do proteÃma as proteÃnas que foram expressas em maior quantidade se localizaram principalmente em pH inferior a 5,5. Comparando a expressÃo da intensidade de spots entre os tratamentos pode-se observar diferenÃa significativa na intensidade de 23 spots em funÃÃo da dieta revelando quinze proteÃnas sendo que desse total, sete foram diferenciadas em funÃÃo do tempo. As proteÃnas identificadas neste experimento podem ser classificadas principalmente como estruturais de organizaÃÃo celular e proteÃÃo ao stresse, bem como com especÃficas funÃÃes metabÃlicas e outras funÃÃes. A adiÃÃo de diferentes fontes de lipÃdeo suplementar influenciou o desempenho produtivo e as caracterÃsticas: rendimento da carcaÃa quente e fria, rendimento e peso da costela o peso e rendimento do trato gastrointestinal cheio, intestino delgado e rendimento do fÃgado em ovinos Santa InÃs em crescimento. A suplementaÃÃo com diferentes fontes lipÃdicas influenciou o perfil de Ãcidos graxos do Longissimus dorsi, de modo que as dietas contendo FCC e Ca-Agcl aumentou a quantidade de CLA neste mÃsculo. A anÃlise do proteoma pode ser um mÃtodo para identificar as proteÃnas marcadoras de prediÃÃo da qualidade da carne. Nesse estudo, as proteÃnas identificadas sÃo altamente relevantes para o processo de maciez da carne, sendo que o processo de maturaÃÃo da carne à complexo e envolve proteÃnas em diferentes processos biolÃgicos. O tipo de alimentaÃÃo teve maior influÃncia sobre as proporÃÃes dos ÃrgÃos responsÃveis pela digestÃo e absorÃÃo de nutrientes.
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Massey, Christopher L. "THE EFFECTS OF QUORUM SENSING AND TEMPERATURE ON THE SOLUBLE PROTEOME OF VIBRIO SALMONICIDA." DigitalCommons@CalPoly, 2016. https://digitalcommons.calpoly.edu/theses/1603.
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Vibrio salmonicida causes cold-water vibriosis in salmon populations around the world and causes financial damage to fisheries designed to farm these salmon. Very little is known about the physiology of how V. salmonicida causes disease and measures to contain vibriosis are restricted to either vaccinating individual fish against disease or administering antibiotics when an outbreak is detected. These procedures are costly and increase the risk for selection of antibiotic-resistant V. salmonicida strains. A recent reoccurrence of outbreaks in Norwegian fisheries provided incentive to better understand the virulence mechanisms of V. salmonicida. In this thesis, a proteomic approach was used to identify proteins that were differentially expressed when cells were grown in vitro under simulated virulence conditions (i.e. 5˚C and in the presence of exogenously supplied autoinducer 3-oxo-hexanoyl-homoserine lactone). Some examples of proteins with significantly altered expression that stood out at as homologs of potential virulence factors were: an exported serine protease DegQ, a multi-drug transporter HlyD, and an outer membrane protein OmpU. The proteomic approach allowed us to identify large numbers of proteins that are expressed by V. salmonicida, facilitating hypothesis-driven research in order to support possible roles for some of these proteins in virulence
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Kimzey, Michael John. "Identification, Characterization, and Quantification of Dicarbonyl Adducts in the Plasma Proteome in Type-2 Diabetes." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/145123.
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Glyco-oxidation is linked to the pathophysiology of diabetes and diabetic complications. The process of glyco-oxidation generates reactive dicarbonyls, which form adducts on arginine residues in distributions throughout the proteome that are site-specific depending on the protein microenvironment. Dicarbonyl adducts are thus markers for glyco-oxidative stress. Various approaches using mass spectrometry permits the identification, localization, and quantification of these dicarbonyl adducts. Using MG as a model dicarbonyl, a shotgun proteomics approach identified the sites for modification of major plasma proteins. Thirty five sites on seven abundant plasma proteins were found, and investigation into the microenvironment surrounding the target arginine sites revealed a neighboring charged residue motif where adjacent residues were either negatively or positively charged. One of the sites identified was R257 in HSA, which is located in the important drug binding site I. We validated drug site I as a target for MG modification by the adaptation of two assays to monitor the effect of MG modification. MG significantly decreases the rate of hydrolysis of PGE2 in drug site I, and induces the displacement of prodan from drug site I. Molecular modeling of warfarin docking at drug site I with the MG-modified R257 resulted in significantly decreased binding and change in binding orientation. The oxidation products of susceptible residues methionine, tryptophan, and cysteine were evaluated using MRM of oxidized HSA peptides. Oxidation of methionine gave the M+16 single oxidized product, and M329 in HSA was the most responsive site. Oxidation of the sole W214 tryptophan produced the W+32 double oxidation product, and oxidation of C34 produced the C+48 triple oxidation product. MG, 3DG, and glucosone were evaluated for propensity to modify 12 HSA sites based on MRM of dicarbonyl modified HSA. Dicarbonyl modification was independent of arginine solvent accessibility. In a clinical study using nephropathy as an endpoint, sites of oxidation and modification of HSA by MG, 3DG, and glucosone were quantified by MRM. The most important variable among diabetic subjects was metformin use, and subjects taking metformin had significantly reduced markers for glyco-oxidation. These findings may be useful in the development of new diabetes therapies that aim to ameliorate glyco-oxidative stress.
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Sun, Jinchun. "A MASS SPECTROMETRY-BASED STUDY OF SERUM BUTYRYLCHOLINESTERASE INHIBITION FROM PESTICIDE EXPOSURE AND ORGANOPHOSPHATE PESTICIDE-INDUCED PROTEOME ALTERATION." UKnowledge, 2006. http://uknowledge.uky.edu/gradschool_diss/290.
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Pesticides including organophosphates (OPs) and carbamates (CBs) are widelyused to control undesirable pests. These compounds are neurotoxic and inhibithydrolysis of the neurotransmitter acetylcholine by acetylcholinesterase. Public healthconcerns have increased with the escalating usage of pesticides. Reliable monitoringprograms are required to detect and quantify pesticide exposure, as well as to promotean understanding of their neurotoxic properties. In this dissertation, both theanticholinergic (Part I) toxicity and neurotoxicity in neuroblastoma cells (Part II) ofpesticides were explored using mass spectrometry (MS). The high sensitivity andhigh-throughput of this technique renders it well-suited for proteomics analysis.Part I describes the study of butyrylcholinesterase (BChE) inhibition resultingfrom OP and CB exposure. The main hypothesis of Part I is that the specialmodification of BChE can provide the origin and extent of pesticide exposure. A novelmethod for detection and quantification of pesticide exposure was designed using aproteomics approach and equine BChE (eBChE) as a model system. The methodologyfeatured detection and analysis of phosphorylated or carbamylated peptides at theactive site serine residue. The developed technique was successfully applied towardsthe study of human BChE (hBChE) inhibition in vitro and in serum samples. Aspecially designed affinity column enabled an isolation of BChE from serum. EnrichedBChE was subjected to enzymatic digestion by a novel on-bead double digestionprotocol. LC/MS/MS was employed to produce a calibration system for the analysis ofhBChE inhibition, which was then applied towards quantification of the enzyme.Part II describes a proteomic study of the neurotoxicity in neuroblastoma cellscaused from chlorpyrifos (CPF), an organophosphate pesticide. The concerns of CPFexposure to pregnant women, infants and children are increasing due todevelopmentally neurotoxic effects of this chemical. The main hypothesis of Part II isthat CPF can cause protein alterations and these altered proteins can be detected usingproteomics. Systematic studies at subcellular levels evaluated proteome changes inSH-SY5Y cells exposed to CPF. Two-dimensional gel electrophoresis (2DE) wasapplied with MALDI-TOF-MS to analyze differential protein expression. Thirty sevencommon unique altered proteins were identified, which play important roles inmetabolic pathway.
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Arivalagan, immanuel Jaison Rathina Raj. "Insights from shell proteome : biomineralization control and environmental adaptation in bivalves." Thesis, Paris, Muséum national d'histoire naturelle, 2017. http://www.theses.fr/2017MNHN0026/document.
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Le processus de biominéralisation confère aux organismes qui le développent une valeur adaptative. La coquille carbonatée des mollusques intègre les fonctions de protection biomécanique à différentes échelles. La coquille résulte de l'association de composés inorganiques et d'une matrice organique protéique, médiatrice du contrôle biologique de la minéralisation. L'analyse du protéome de la coquille chez 4 espèces de bivalves met en évidence deux patrons fonctionnels et leur degré de conservation phylogénétique : l'un lié au contrôle de la minéralisation stricto sensu ; l'autre à la protection immune. L'étude de populations vivant à l'état naturel en mer Baltique, dont les eaux présentent localement de fortes variations ioniques montre que le protéome intègre également l'impact de conditions environnementales limitantes. L'anthropocène impose un rythme adaptatif pressant aux organismes et la modification acido-basique des eaux océaniques est susceptible d'impacter sensiblement les organismes calcifiants. La signature de mécanismes adaptatifs du contrôle biologique de la biominéralisation se traduit dans le protéome de la coquille. Les implications sont particulièrement signifiantes dans un contexte d'intérêt de développement aquacole grandissantIn this study, the SMPs from four commercially important and divergent bivalve species crassostrea gigas (pacific oyster), Mya truncata (soft shell clam), Mytilus edulis (blue mussel) and Pecten maximus (king scallop) were extracted and analysed using standardized extraction protocol and proteomic pipeline. This enables us to identify critical elements of basic biomineralization tool kit for calcification process irrespective of their shell morphology, mineralogy and microstructure. In addition, it enables the identification of SMPs that are specific to calcite and aragonite mineralogies. The signifiant numbers of SMPs found species-specific were hypothesized as adaptation to their modus vivendi. In fact, the latter proteins possess immunity-related functions and fit into specific pathway, phenoloxidase, suggesting their role in defense against pathogen. The comparative study of shell proteome of mussels living in full marine condition, North Sea and the Iow saline Baltic Sea showed the modulation of the SMPs that constitute the basic biomineralization tool kit. Higher modulation of chitin related proteins and non-modulated protein such as carbonic anhydrase, EGF and fibronectin domain containing proteins points out the impaired scaffold and mineral nucleation process in Baltic mussel. The modulation of immunity related proteins denote the influence of biotic components. These investigations show the functional diversity of SMPs and their roles beyond shell formation in the bivalvesand put forth the idea that shell is dynamic, endowed with both biochemical and mechanical protection
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Strandberg, Linnéa. "Isolation of the native chloroplast proteome from plant for identification of protein-metabolite interactions." Thesis, KTH, Proteinvetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-301783.
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För att kunna livnära en växande population behöver avkastningen på skördar öka. En lösning på dettaär att optimera plantornas fotosyntes, vilket innefattar förbättrad koldioxidfixering. För att lyckas meddet krävs kunskap i hur reglering av nyckelproteiner i kloroplasten går till. Syftet med detta projekt är identifiera möjliga reglerande protein-metabolitinteraktioner i Arabidopsis thaliana. Målproteinerna ärde 11 enzymerna i Calvin-Benson-Basshamcykeln. Metaboliterna som testas är 3PGA, ATP, FBP, GAP, vilka är mellan produkter eller kofaktorer i cykeln; 2PG, som är en produkt av en konkurrerande reaktion i cykeln; och slutligen G6P, citrat och sackaros, vilka är centrala metaboliter i andra viktiga reaktioner i cellen. Före experimenten med Arabidopsis testades protokollen med spenat. Som ett första steg isolerades kloroplasterna från blad. När intakta kloroplaster verifierats extraherades proteinerna. Inter-aktioner mellan metaboliterna och proteinerna analyserades med en metod kallad limited proteolysis-small molecule mapping. Denna teknik, vilken kombinerar begränsad proteolys med masspektrometri, detekterade flertalet protein-metabolit interaktioner. I Arabidopsis uppvisade alla enzym förutom FB-Pase, PPE och TIM minst en interaktion. I spenat sågs interaktioner med FBA, GAPDH, PGK, PRK, RuBisCO, TIM och TK. Resultaten visar möjliga reglerande interaktioner, vilka skulle kunna användasför att identifiera flaskhalsar i kolfixeringen. Denna kunskap kan i sin tur utnyttjas för att öka flödet i Calvin-Benson-Basshamcykeln och därigenom förbättra växters koldioxidfixering.In order to feed a growing population, the crop yield needs to be increased. One way to do this is to optimise the photosynthetic activity in the plant, which includes improvement of carbon fixation. To succeed with this, knowledge of the regulation of key proteins in the chloroplast is required. The aim of this project is to identify possible regulatory protein-metabolite interactions in chloroplasts from Arabidopsis thaliana. The target proteins are the 11 enzymes of the Calvin-Benson-Bassham cycle. The metabolites of interest are 3PGA, ATP, FBP, GAP, which are intermediates or co-factors of the cycle;2PG, which is a product of a competing reaction in the cycle; and finally G6P, citrate and sucrose, which are central metabolites in other vital reactions in the cell. Before the experiments with Arabidopsis, spinach was used as a test organism to evaluate the proposed protocols. First, chloroplasts were isolatedfrom leaves. When the integrity of the chloroplasts had been validated, the proteins were extracted. Metabolic interactions with the extracted proteins were analyzed with limited proteolysis-small molecule mapping. This method, which combines limited proteolysis with mass spectrometry, detected severalprotein-metabolite interactions. In Arabidopsis, all enzymes except for FBPase, PPE and TIM had atleast one interaction. In spinach, interactions were seen with FBA, GAPDH, PGK, PRK, RuBisCO,TIM and TK. The results highlight potential regulatory events, which could be used to target bottlenecks in carbon fixation. This could provide a pathway to increase the flux in the Calvin-Benson-Bassham cycle, and thereby improve carbon fixation in plants.
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So, Wing-yan, and 蘇詠欣. "Proteome and gene expression analysis in white adipose tissue of diet-induced obese mice." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39367435.
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