Academic literature on the topic 'Label-free quantitative proteomic'
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Journal articles on the topic "Label-free quantitative proteomic"
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Ankney, J. Astor, Adil Muneer, and Xian Chen. "Relative and Absolute Quantitation in Mass Spectrometry–Based Proteomics." Annual Review of Analytical Chemistry 11, no. 1 (June 12, 2018): 49–77. http://dx.doi.org/10.1146/annurev-anchem-061516-045357.
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Mass spectrometry–based quantitative proteomics is a powerful tool for gaining insights into function and dynamics of biological systems. However, peptides with different sequences have different ionization efficiencies, and their intensities in a mass spectrum are not correlated with their abundances. Therefore, various label-free or stable isotope label–based quantitation methods have emerged to assist mass spectrometry to perform comparative proteomic experiments, thus enabling nonbiased identification of thousands of proteins differentially expressed in healthy versus diseased cells. Here, we discuss the most widely used label-free and metabolic-, enzymatic-, and chemical labeling–based proteomic strategies for relative and absolute quantitation. We summarize the specific strengths and weaknesses of each technique in terms of quantification accuracy, proteome coverage, multiplexing capability, and robustness. Applications of each strategy for solving specific biological complexities are also presented.
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Abdallah, Cosette, Eliane Dumas-Gaudot, Jenny Renaut, and Kjell Sergeant. "Gel-Based and Gel-Free Quantitative Proteomics Approaches at a Glance." International Journal of Plant Genomics 2012 (November 20, 2012): 1–17. http://dx.doi.org/10.1155/2012/494572.
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Two-dimensional gel electrophoresis (2-DE) is widely applied and remains the method of choice in proteomics; however, pervasive 2-DE-related concerns undermine its prospects as a dominant separation technique in proteome research. Consequently, the state-of-the-art shotgun techniques are slowly taking over and utilising the rapid expansion and advancement of mass spectrometry (MS) to provide a new toolbox of gel-free quantitative techniques. When coupled to MS, the shotgun proteomic pipeline can fuel new routes in sensitive and high-throughput profiling of proteins, leading to a high accuracy in quantification. Although label-based approaches, either chemical or metabolic, gained popularity in quantitative proteomics because of the multiplexing capacity, these approaches are not without drawbacks. The burgeoning label-free methods are tag independent and suitable for all kinds of samples. The challenges in quantitative proteomics are more prominent in plants due to difficulties in protein extraction, some protein abundance in green tissue, and the absence of well-annotated and completed genome sequences. The goal of this perspective assay is to present the balance between the strengths and weaknesses of the available gel-based and -free methods and their application to plants. The latest trends in peptide fractionation amenable to MS analysis are as well discussed.
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Silva, Wanderson M., Cassiana S. Sousa, Leticia C. Oliveira, Siomar C. Soares, Gustavo F. M. H. Souza, Guilherme C. Tavares, Cristiana P. Resende, et al. "Comparative proteomic analysis of four biotechnological strainsLactococcus lactisthrough label-free quantitative proteomics." Microbial Biotechnology 12, no. 2 (October 19, 2018): 265–74. http://dx.doi.org/10.1111/1751-7915.13305.
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Zhang, Yan, and Ioannis P. Nezis. "Label-free quantitative proteomic analysis of adult Drosophila heads." STAR Protocols 3, no. 4 (December 2022): 101830. http://dx.doi.org/10.1016/j.xpro.2022.101830.
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Burch, Andrew R., Cody W. Yothers, Michelle R. Salemi, Brett S. Phinney, Pramod Pandey, and Annaliese K. Franz. "Quantitative label-free proteomics and biochemical analysis of Phaeodactylum tricornutum cultivation on dairy manure wastewater." Journal of Applied Phycology 33, no. 4 (May 27, 2021): 2105–21. http://dx.doi.org/10.1007/s10811-021-02483-3.
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AbstractMicroalgae cultivation on wastewater offers the dual benefit of lowering costs for feedstock production with simultaneous wastewater remediation. This study utilized biochemical and quantitative label-free proteomic approaches to evaluate the growth and proteomic response for diatom Phaeodactylum tricornutum cultivated on flushed dairy manure wastewater (DMW). Comparing several DMW dilutions (up to 60% DMW diluted in seawater) with a synthetic seawater medium indicates that biomass and lipid yields correlate with the starting nitrogen content of the DMW dilution. Phaeodactylum tricornutum cultivated on DMW exhibits elevated levels of polyunsaturated fatty acids (PUFAs), particularly docosapentaenoic acid (DPA, 22:5 n-3). Proteomic analysis revealed alterations in the regulations of proteins associated with protein metabolism, cellular signaling, transcription and translation, protein trafficking, and oxidative stress management pathways when comparing P. tricornutum cultivation on diluted DMW versus synthetic media, thus providing insights into how P. tricornutum reorganizes its proteome in response to a complex wastewater source. Graphical abstract
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Karpiński, Adam Aleksander, Julio Cesar Torres Elguera, Anne Sanner, Witold Konopka, Leszek Kaczmarek, Dominic Winter, Anna Konopka, and Ewa Bulska. "Study on Tissue Homogenization Buffer Composition for Brain Mass Spectrometry-Based Proteomics." Biomedicines 10, no. 10 (October 2, 2022): 2466. http://dx.doi.org/10.3390/biomedicines10102466.
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Mass spectrometry-based proteomics aims to study the proteome both qualitatively and quantitatively. A key step in proteomic analysis is sample preparation, which is crucial for reliable results. We investigated the effect of the composition of the homogenization buffer used to extract proteins from brain tissue on the yield of protein extraction and the number and type of extracted proteins. Three different types of buffers were compared—detergent-based buffer (DB), chaotropic agent-based buffer (CAB) and buffer without detergent and chaotropic agent (DFB). Based on label-free quantitative protein analysis, detergent buffer was identified as the most suitable for global proteomic profiling of brain tissue. It allows the most efficient extraction of membrane proteins, synaptic and synaptic membrane proteins along with ribosomal, mitochondrial and myelin sheath proteins, which are of particular interest in the field of neurodegenerative disorders research.
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Arnold, Georg J., and T. Frohlich. "Dynamic proteome signatures in gametes, embryos and their maternal environment." Reproduction, Fertility and Development 23, no. 1 (2011): 81. http://dx.doi.org/10.1071/rd10223.
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Comprehensive molecular analysis at the level of proteins represents a technically demanding, but indispensable, task since several post-transcriptional regulation mechanisms disable a valid prediction of quantitative protein expression profiles from transcriptome analysis. In crucial steps of gamete and early embryo development, de novo transcription is silenced, meaning that almost all macromolecular events take place at the level of proteins. In this review, we describe selected examples of dynamic proteome signatures addressing capacitation of spermatozoa, in vitro maturation of oocytes, effect of oestrous cycle on oviduct epithelial cells and embryo-induced alterations to the maternal environment. We also present details of the experimental strategies applied and the experiments performed to verify quantitative proteomic data. Far from being comprehensive, examples were selected to cover several mammalian species as well as the most powerful proteomic techniques currently applied. To enable non-experts in the field of proteomics to appraise published proteomic data, our examples are preceded by a customised description of quantitative proteomic methods, covering 2D difference gel electrophoresis (2D-DIGE), nano-liquid chromatography combined with tandem mass spectrometry, and label-free as well as stable-isotope labelling strategies for mass spectrometry-based quantifications.
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Kohli, Priyanka, Malte P. Bartram, Sandra Habbig, Caroline Pahmeyer, Tobias Lamkemeyer, Thomas Benzing, Bernhard Schermer, and Markus M. Rinschen. "Label-free quantitative proteomic analysis of the YAP/TAZ interactome." American Journal of Physiology-Cell Physiology 306, no. 9 (May 1, 2014): C805—C818. http://dx.doi.org/10.1152/ajpcell.00339.2013.
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The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.
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Di Luca, Alessio, Andrea Ianni, Francesca Bennato, Michael Henry, Paula Meleady, and Giuseppe Martino. "A Label-Free Quantitative Analysis for the Search of Proteomic Differences between Goat Breeds." Animals 12, no. 23 (November 29, 2022): 3336. http://dx.doi.org/10.3390/ani12233336.
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The intensification and standardization of livestock farming are causing a decline in the number of animal breeds in many species, such as the goat. The availability of more studies on the potentiality of goat breeds could raise awareness of their importance, conservation and productive possibilities. Label-free quantitative analysis was applied in this study to investigate the proteomic differences between the autochthon Teramana and Saanen goats that could be useful for defining peculiar features of these breeds. A total of 2093 proteins were characterized in the muscle exudate proteome of the Teramana and Saanen breeds. A total of 41 proteins clearly separated the two breeds. Eukaryotic initiation factor proteins and aldehyde-dehydrogenase 7 family-member A1 were up-regulated in the autochthon breed and associated with its resilience, whereas catalase was down-regulated and associated with lower muscular mass. This study is the most detailed report of goat muscle proteome. Several differentially regulated proteins between the two breeds were identified, providing insights into functional pathways that define this organism and its biology.
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Mosley, Amber L., Laurence Florens, Zhihui Wen, and Michael P. Washburn. "A label free quantitative proteomic analysis of the Saccharomyces cerevisiae nucleus." Journal of Proteomics 72, no. 1 (February 2009): 110–20. http://dx.doi.org/10.1016/j.jprot.2008.10.008.
Full textDissertations / Theses on the topic "Label-free quantitative proteomic"
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Cummings, Rebecca. "Development and application of label free quantitative proteomic methods." Thesis, University of Liverpool, 2012. http://livrepository.liverpool.ac.uk/8313/.
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The aim of this Ph.D. was to develop advanced methods for quantitative proteomics and use these methods to investigate the presence of protein biomarkers of sperm performance, differential expression of sperm membrane proteins and differential expression of E.coli proteins. Quantitative analysis of E.coli generated analytical samples that were analysed with multiple mass spectrometers and with multiple software packages. Through these samples an optimal label free quantitative proteomic workflow was generated and software was thoroughly tested to determine the optimal software to be used for data analysis on varying biological questions. Identification of protein(s) that correlate with increased or decreased fertility would be economically beneficial. Currently semen samples are subject to quality control where general movement and morphological defects are studied, but this does not always correlate with the ejaculate passing a post cryopreservation quality control check or that specific bull generating offspring. Identification of a protein or set of proteins with abundance variation in bulls of known high or low fertility would allow lower fertility bulls to be removed from the breeding programme at an early age, reducing rearing costs, and would allow longitudinal health monitoring of individual bulls. Discovery of differentially expressed proteins in the membrane of sperm with the X or Y chromosome would allow the generation of a method to separate the two sperm populations. This will be beneficial as most livestock farmers would prefer offspring of a specific sex, either to sell or replenish animal stock. Quantitative analysis of proteins present in bovine seminal plasma led to the identification and quantitative comparison of the seminal plasma proteins present in two breeds of bull, Holstein and Belgian Blue and a quantitative comparison of the seminal plasma from two domestic farm animal species, bovine and porcine. Intra species comparisons determined no quantitative variation between the two breeds, while the inter species comparison determined variation between the proteins present in both species seminal plasma and the corresponding amounts of proteins present in both species. A quantitative comparison was performed to determine the expression of proteins from two strains of E.coli, a wild type strain (MG1655) and a genome depleted strain (MDS66), this led to the confirmation of gene deletions in the genome depleted strain due to their lack of protein products in mass spectrometric analysis, and the identification of proteins that were differentially expressed due to pleiotropic effects of these genome deletions. To investigate the proteins expressed in the sperm membrane a mass spectrometer compatible enrichment method was generated and membrane proteins were identified, quantified and compared between sperm expressing X and Y chromosomes. This study did not lead to the determination of any proteins with differential expression in the X or Y bearing sperm.
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Paiva, Ana Luiza Sobral. "Biochemical responses of bean-to-string [Vigna unguiculata L. (Walp.)] to salt stress and infection by severe mosaic of cowpea (CPSMV) revealed by quantitative proteomics dial free." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=13591.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoAs sessile organisms, plants are exposed to a plethora of environmental stresses to which they must respond to maintain efficient growth and survival. Therefore, in order to improve our understanding on the complex mechanisms involved in the cowpea response to salt stress and to a compatible interaction with the cowpea severe mosaic virus (CPSMV), we used a label-free quantitative proteomic approach to identify the salt and virus responsive proteins in the leaves of the Pitiuba (CE-31) cultivar. The proteins extracted from the leaves (control and treated) 2 and 6 days post-treatment only with salt (DPS), only infected with CPSMV (DPV) or both of them (DPSV) were analyzed using mass spectrometry. At 2 DPS, 350 proteins with at least two-fold differences in abundance, in comparison with controls, were differentially accumulated in the leaves of the salt-treated (80% up and 20% down-accumulated), 281 at 2DPV (25% up and 75% down-accumulated) and 321 at 2 DPSV (45% up and 55% down-accumulated) plants. At 6 DPS, 350 proteins were differentially accumulated in the leaves of the salt-treated (90% up and 10% down-accumulated), 225 at 6 DPV (80% up and 20% down-accumulated) and 315 at 6 DPSV (94% up and 6% down-accumulated) plants. The qualitative analysis showed biochemical differences when the cowpea plants were challenged concurrently with both stresses. To cope with salinity, cowpea increased the abundance of proteins directly involved with the salt tolerance mechanisms. The results indicated that the CPSMV induce the down-accumulating of several proteins to invade and spread in host at early infection period (2 DPV), but at 6 DPV plant can induce accumulation of diverse proteins related with defense, although these strategies canât avoid the negatives effects of disease. When exposed simultaneously to salt/CPSMV stresses, a balance in protein accumulation involved in many biological process. This is the first work employing this approach in cowpea and providing evidences of the plant biochemical mechanisms involved in the responses of cowpea to these stresses.
Como organismos sÃsseis, as plantas sÃo expostas a uma variedade de estresses ambientais aos quais devem responder para sobreviverem e se desenvolverem. A fim de melhorar a nossa compreensÃo sobre os mecanismos complexos envolvidos na resposta do feijÃo-de-corda ao estresse salino e na interaÃÃo compatÃvel com o vÃrus do mosaico severo do caupi (CPSMV), foi utilizada uma abordagem proteÃmica quantitativa, livre de marcaÃÃo, para identificar proteÃnas, responsivas a essess estresses em folhas de feijÃo-de-corda, cv. CE-31. As proteÃnas extraÃdas a partir de folhas primÃrias, 2 e 6 dias apÃs o tratamento sà com o sal (DPS), somente infectadas (DPV), ou sob aÃÃo combinada dos dois (DPSV) foram analisadas, usando espectrometria de massas e comparadas com grupo controle. No 2 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (80% aumentaram em abundÃncia e 20% diminuÃram), no 2 DPV 281 (25% aumentaram em abundÃncia e 75% diminuÃram) e no 2 DPSV 321 (45% aumentaram em abundÃncia e 55% diminuÃram). Jà no 6 DPS, foram identificadas 350 proteÃnas diferencialmente acumuladas (90% mostraram aumento em abundÃncia e 10% diminuiÃÃo), no 6 DPV 225 (80% aumentaram em abundÃncia e 20% diminuÃram) e no 6 DPSV 315 proteÃnas(94% aumentaram em abundÃncia e 6% diminuÃram). Para lidar com a salinidade, o cv. CE-31 aumentou a abundÃncia de proteÃnas envolvidas diretamente com os mecanismos de tolerÃncia ao sal. Em relaÃÃo à infecÃÃo da planta pelo CPSMV, os resultados obtidos indicaram que o vÃrus induz reduÃÃo na abundÃncia de vÃrias proteÃnas nos tempos iniciais de infecÃÃo, provavelmente favorecendo a invasÃo e propagaÃÃo na planta, mas, no 6 DPSV, a planta recupera sua capacidade de acionar mecanismos de defesa, embora esses jà nÃo sejam mais efetivos para evitar o estabelecimento da doenÃa viral. Durante exposiÃÃo simultÃnea da planta ao sal e ao vÃrus, ocorreu um equilÃbrio entre o aumento e diminuiÃÃo em abundÃncia de proteÃnas envolvidas em diversos processos metabÃlicos. Esse trabalho à pioneiro nessa abordagem em feijÃo-de-corda e fornece evidÃncias dos mecanismos bioquÃmicos envolvidos nas resposta da planta a esses estresses.
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Bertaccini, Diego. "Advances in analytical methodologies for the characterization and quantification in proteomic analysis." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF043/document.
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L’objectif de cette thèse était de développer et d’optimiser de nouvelles méthodologies et approches analytiques afin d’améliorer le potentiel de l’analyse protéomique pour les études biologiques.La première partie de ce travail est consacrée à la détermination massive et exacte de la position N-Terminale des protéines (N-Terminome). Pour cela, nous avons utilisé et développé une approche basée sur une dérivation N-Terminale au TMPP. Cette méthodologie de marquage de la position N-Terminale a permis d’aborder l’étude des clivages protéolytiques des protéines exportées par le parasite P. falciparum (pathogène de la malaria) dans le globule rouge.Afin de permettre une exploitation automatique à haut débit des données de MS/MS, nous avons élaboré une nouvelle méthodologie (dénommée dN-TOP). Celle-Ci repose sur l’utilisation de TMPP portant des isotopes stables et permet ainsi d’accéder à la détermination des positions N-Terminales pour des études de N-Terminome à large échelle.La seconde partie est dédiée aux développements de différentes stratégies analytiques de quantification, aussi bien au niveau peptidique qu’au niveau protéique, appliquées à une série de problématiques biologiques. Ces optimisations ont été réalisées dans le contexte de l’étude des complexes protéiques, du dosage de prion par SRM, de quantification des glycations d’anticorps monoclonaux thérapeutiques et de l’hémoglobine HbA2 pour la standardisation des méthodes de référenceThe objective of this Ph.D. thesis was to develop and optimize new methodologies and analytical approaches to improve the potential of the mass spectrometry based proteomics.The first part of this work focused on the development of the N-Termini proteomics. This topic was addressed with a specific N-Termini chemical derivatization based on TMPP. We have shown that our method allowed both specific N-Terminomics and classical proteomics studies in the same experiment.This N-Terminus methodology was applied to study the proteolytic cleavages of the exported proteins in P. falciparum, a parasite responsible for the malaria.In order to automatize the complex and tedious informatics processsing of the MS/SM data of ourTMPP based N-Terminomics method, we have introduced a new approach (named dN-TOP), based on the use of a stable isotope labeled TMPP which made now N-Terminome proteomics compatible with high throughput studies.The second part addresses quantitative aspects of proteomics. It describes the optimization of quantitative methods at the peptide level or at the protein level for five different proteomic studies in the context of protein complex subunits, targeted SRM based prion, quantification of monoclonal antibodies glycation and hemoglobin HbA2 for reference measurement methods standardization
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Zaccarin, Mattia. "Setting up of an innovative procedure for redox proteomics: and its application for definition of the redox status of cells with high metastatic potential." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423382.
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BACKGROUND: The cysteine (Cys) proteome includes 214.000 Cys with thiol and other forms. Of these, only a relatively small subset functions in cell signalling. Redox-active Cys are more susceptible to oxidation, and their oxidized form is more susceptible to reduction. Specific proteomic techniques are required to identify these modifications and to study their regulation in different cell processes that are collectively known as redox proteomics. Thus, it is of interest to be able to identify both the proteins and the cysteine residues affected, and to quantify the extent of the modification involved.The quantification of differences between two or more physiological states of a biological system is among the most challenging technical tasks in proteomics: liquid chromatography coupled to mass spectrometry (LC-MS) based quantification methods have gained increasing robustness and reliability over the past five years. Many authors still share a view of redox signalling in which the fate of the cell is dependent mainly on the intensity and duration of pro-oxidant stimulus: here we sustain the involvement of an equilibrium encompassing the action of both nucleophiles and electrophiles at the same time.
AIM: The dual aim of my PhD work has been both to develop suitable methodology to identify and quantify redox-active proteins in complex samples and to apply it to the study of a cellular model of breast cancer (MCF10A) engineered to reproduce malignancy.
METHODS: In order to pursue this aim, I took advantage of an approach integrating differential chemical sample labelling (non-isotopic) with Cys reactive probes (NEM, IAM, HPDP) and chromatographic purification of redox-sensitive proteins, with subsequent LC-MS/MS analysis and computational data handling for OpenMS-based label-free quantification. All the steps of this methodology have been developed and validated in close collaboration with experts from both the biochemistry and bioinformatics field.
RESULTS: We obtained an efficient cost-effective and isotopes-free methodology to characterize the redoxome in complex protein samples. Application of our quantification protocol to benchmark dataset leads to 100% correct estimates of under/over expression of the protein moiety. Application of the methodology to the breast cancer cellular model lead to identification of more than 300 proteins and allowed us to group-up unchanged and differentially oxidized redox-sensitive proteins in the more malignant cells in respect to their less aggressive counterpart.
CONCLUSION: Despite the commonly accepted association between cancer and higher oxidative-stress, this study links higher breast cancer cells malignancy to a finely tuned dynamic equilibrium in which selected protein targets are oxidized in the context of a more reduced cell environment. Preliminary results point at the enzyme G6PDH as a crucial regulator of this redox process.STATO DELL’ARTE: Il proteoma include 214.000 cisteine in forma di gruppi tiolici liberi od altra forma. Di queste, solamente un insieme relativamente ristretto ha un ruolo nella mediazione di segnali cellulari. Tali cisteine, attive dal punto di vista dell’ossido-riduzione, sono più sensibili all’ossidazione e la loro forma ossidata è più facilmente riducibile. Sono dunque necessarie specifiche tecniche di proteomica, globalmente indicate con il termine proteomica delle ossido-riduzioni, per identificare tali modifiche e studiarne la regolazione in diversi processi cellulari. Risulta quindi determinante la capacità di identificare sia le proteine che i residui coinvolti e di quantificarne il grado di modificazione. E proprio la quantificazione delle differenze tra due o più stati di un sistema biologico, si colloca tra gli obiettivi tecnicamente più sfidanti della proteomica: nel corso degli ultimi cinque anni, tecniche basate sulla spettrometria di massa associata a cromatografia in fase liquida hanno progressivamente guadagnato affidabilità e robustezza. Molti autori condividono tuttora una visione delle ossido-riduzioni nella mediazione del segnale in cui il destino cellulare dipende principalmente dall’intensità e dalla durata degli stimoli ossidanti: nel presente lavoro si vuole invece sostenere il coinvolgimento di un equilibrio che includa l’azione concomitante sia di specie nucleofile sia di specie elettrofile. OBIETTIVO: Il duplice obiettivo del mio lavoro di Dottorato è stato sia lo sviluppo di una metodologia idonea all’identificazione e quantificazione di proteine, attive dal punto di vista delle ossido-riduzioni, in campioni complessi, sia l’applicazione di tale metodologia allo studio di un sistema cellulare ingegnerizzato di carcinoma mammario (MCF10A) caratterizzato da diversi gradi di malignità. METODI: Al fine di perseguire tale obiettivo ho tratto vantaggio da un approccio che integra la marcatura chimica differenziale (non-isotopica) per mezzo di sonde reattive con i residui di cisteina (NEM, IAM, HPDP) e la purificazione cromatografica delle proteine attive dal punto di vista ossido-riduttivo, alla successiva analisi LC-MS/MS ed elaborazione informatizzata dei dati mediante OpenMS per una quantificazione label-free. Tutti i passaggi di tale metodologia sono quindi stati messi a punto e validati in stretta collaborazione con esperti biochimici e bioinformatici. RISULTATI: E’ stato sviluppato un metodo efficiente ed economico, non basato sull’utilizzo di marcatori isotopici, per la caratterizzazione delle proteine attive dal punto di vista ossido-riduttivo in campioni proteici complessi. L’applicazione del protocollo di quantificazione ad un campione test ha dato il 100% di stime corrette di sovra/sotto-espressione della miscela proteica. L’applicazione del metodo allo studio del modello cellulare di carcinoma mammario ha portato all’identificazione di più di 300 proteine ed ha permesso il raggruppamento di quelle sensibili dal punto di vista ossido-riduttivo in gruppi non differenziali e sovra- o sotto-ossidate nelle cellule più maligne rispetto alla loro controparte meno aggressiva. CONCLUSIONI: Nonostante sia comunemente riconosciuta l’associazione tra fenomeni neoplastici ed uno stress ossidativo, questo studio collega la maggiore malignità di un modello cellulare di carcinoma mammario ad un complesso equilibrio ossido-riduttivo. In questo contesto, specifici bersagli proteici sono ossidati mentre viene mantenuto un ambiente cellulare complessivamente ridotto. Risultati preliminari evidenziano poi l’enzima G6PDH come possibile elemento chiave nella regolazione di tale equilibrio.
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Muller, Leslie. "Développements de méthodes de préparation d’échantillons pour l’analyse protéomique quantitative : application à la recherche de biomarqueurs de pathologies." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF067/document.
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Les stratégies de protéomique quantitative sans marquage sont très attractives dans le domaine de la recherche de biomarqueurs de pathologies. Cependant, elles requièrent une pleine maîtrise du schéma analytique et de sa répétabilité. Plus particulièrement, la préparation d’échantillons nécessite d’être suffisamment répétable pour ne pas impacter la qualité et la fiabilité des résultats. Les objectifs de cette thèse étaient de développer et d’optimiser des méthodes analytiques pour la protéomique quantitative, en particulier pour l’étape de préparation d’échantillons. Ainsi, un protocole innovant, simple, rapide et permettant l’analyse quantitative sans marquage d’un grand nombre d’échantillons avec une haute répétabilité a été développé et optimisé : le « Tube-Gel ». Par ailleurs, des préparations d’échantillons adaptées à différentes matrices biologiques pour la recherche de biomarqueurs ont été élaborées. Les méthodes mises au point et leur application ont permis de proposer des candidats biomarqueurs pour plusieurs pathologies : le glioblastome, les lymphomes B diffus à grandes cellules, et les complications survenant sur les greffons rénauxLabel-free quantitative proteomics strategies are very attractive for diseases biomarkers researches. These approaches require the full control and the repeatability of the analytical workflow. In particular, the sample preparation has to be repeatable enough to ensure the quality and reliability of the results. Objectives of this work were to optimize and develop analytical methods for quantitative proteomics, with a special focus on the sample preparation step. Thus, an innovative, easy and fast protocol allowing the analysis of high sample numbers with high repeatability was developed and further optimized: the “Tube-Gel” protocol. Besides,sample preparations adapted to a variety of biological matrices were developed for the search of biomarkers. The developed methods and their application allowed the identification of potential biomarkers for a variety of diseases: glioblastoma, diffuse large B-cell lymphomas and renal transplants failures
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NUKALA, SARATH BABU. "BIOANALYTICAL AND PROTEOMIC APPROACHES IN THE STUDY OF PATHOLOGIC ECS DYSFUNCTIONALITY, OXIDATIVE STRESS AND THE EFFECTS OF PFKFB3 MODULATORS." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/644236.
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Vascular dysfunction is one of the primary factors in the onset and progression of atherosclerosis and other vascular-related diseases such as acute myocardial infarction (AMI) and chronic thromboembolic pulmonary hypertension (CTEPH). Emerging evidence indicates that pathological blood vessel responses and endothelial dysfunction are associated with metabolic alterations in endothelial cells (ECs). The identification of the insights and causes resulting in dysfunctional ECs is crucial for the understanding of the disease and to the development of new therapeutic tools. Therefore, to study the characteristics of such EC dysfunction in AMI and CTEPH diseases, we aim to determine the protein profile and/or the altered redox status of patient-derived EC lines by using mass spectrometry-based label-free quantitative proteomic and/or, spectrophotometric and fluorometric approaches. Moreover, quantitative proteomic approach was used to explore the underlying mechanisms of 3PO [(3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), PFKFB3 inhibitor] at cellular level. 3PO is a compound able to inhibit the glycolytic flux partially and transiently and to reduce pathological angiogenesis in a variety of disease models.
Bioinformatic and network analyses performed on pathologic HCAEC-AMI cells revealed the alteration of a) metabolism of RNA, b) platelet activation, signaling and aggregation, c) neutrophil degranulation, d) metabolism of amino acids and derivatives, e) cellular responses to stress and, f) response to elevated platelet cytosolic Ca2+ pathways. Similarly, network analysis in pathologic CTEPH-ECs, revealed the differentially regulation of pathways related to a) neutrophil and platelet degranulation, b) metabolism of lipids, amino acids and selenoamino acids, c) response to elevated cytosolic Ca2+, d) detoxification of reactive oxygen species. In addition, the main parameters, indicators of the redox status of both HCAEC-AMI and CTEPH-ECs were significantly increased: advanced oxidation protein products (AOPPs), protein carbonyls (PCO) content, and intracellular reactive oxygen species (ROS). Interestingly, the amount of GSH/GSSG (reduced glutathione/oxidized glutathione) and NADPH/NADP (reduced/oxidized form of nicotinamide adenine dinucleotide phosphate) ratios in dysfunctional ECs were reduced, a clear indication of oxidative stress involvement in both the pathological ECs. Finally, 3PO has multiple targets in the ECs, targeting mitochondrial inner membrane and it inhibits the important cellular pathways including the tricarboxylic acid cycle, the mitochondrial respiratory chain, and vasculogenesis, that may be useful for understanding the inhibitory effect of 3PO on EC proliferation and migration. Therefore, the present data suggest a potential application of this molecule as a starting point in designing novel molecules to prevent diseases where inflammatory reactions are involved, such as in atherosclerosis, cancer or neurodegenerative diseases.
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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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Mackay, Katherine. "A comparative study of analysis methods in quantitative label free proteomics." Thesis, University of Liverpool, 2015. http://livrepository.liverpool.ac.uk/2050359/.
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The large amounts of data generated by modern proteomics experiments necessitates the use of software pipelines to conduct the bulk of the post-processing. While many software packages (both commercial and open-source) are available to perform some or all of the necessary post-processing steps, it is usual for each research group to use only the instrumentation and software packages with which they are most familiar and/or which are available to analyse their unknown data. The intention of the studies presented within this thesis was to assess the correlation between the experimental results obtained when; - a single result dataset is obtained and post-processed in parallel using four separate software pipelines - a single sample is analysed on two different mass spectrometers and post-processed in parallel and; - when different identification thresholds are applied to a dataset prior to parallel quantitation of the resultant data sets Correlation between different mass spectrometry instruments was assessed and found to yield high r values, especially at the protein level, and was also found to improve following the application of abundance thresholds, however the result of applying score thresholds was unpredictable. The use of manual FDR thresholds prior to importing data into Progenesis LC-MS yielded interesting results, which suggest that a threshold of 1% peptide FDR and 1 or 2% protein FDR is most effective in terms of yielding accurate ratios while maintaining acceptable sensitivity. In addition, a consensus method is suggested to utilise the results from multiple software pipelines in order to increase sensitivity and reduce the FDR, through the use of the QPROT tool and manual post-processing.
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Jarnuczak, Andrew. "Mass spectrometry-based quantitative proteomics applied to the analysis of Saccharomyces cerevisiae heat stress response and chaperone deletion strains." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/mass-spectrometrybased-quantitative-proteomics-applied-to-the-analysis-of-saccharomyces-cerevisiae-heat-stress-response-and-chaperone-deletion-strains(c653915b-70fa-44d7-9bb7-6e7965349ff0).html.
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In the last decade omics technologies enabled detailed and system-wide analysis of complex biological samples. Genomics, transcriptomics and metabolomics all benefited tremendously from technological advances in their respective fields. Proteomics was revolutionised by mass spectrometry, which allowed simultaneous identification of thousands of proteins in cells, tissues and organisms. And this mainly qualitative revolution, quickly turned quantitative. This work had two main objectives. Firstly, to apply the state of the art instrumentation, data analysis and bioinformatics methods to better our understanding of basic cell biology in a model organism Saccharomyces cerevisiae. Specifically, to quantitatively describe the effects of perturbations, such as adverse environmental conditions or chaperone gene deletions, on protein abundances in the cell. Additionally, it was aimed to demonstrate and evaluate the ability of a new timeof-flight mass spectrometer to perform large-scale absolute quantification. First, it was found that yeast cells are remarkably robust to deletions of major chaperone hub proteins (Ssa1p or Ssb1p deletions). This ability was attributed to network structure and redistribution of folding workload among other related chaperones rather than simple functional redundancy. Second, to build on the first set of results, a detailed time resolved description of yeast proteome dynamics in response to heat stress was provided for the wild type and Ssb1p chaperone mutant strains. In this study, for the first time in the literature, temporal expression patterns of many hallmark heat shock proteins were elucidated. Globally, a slow and sustained proteome remodelling or 'buffering' was revealed in both strains. However, it was also shown that the cells knocked out for the Ssb1p chaperone respond to heat in a distinctly different manner to the wild type strain. Finally, consistent and reproducible absolute quantification of multiple yeast proteomes was demonstrated using a new commercial time-of-flight mass spectrometer with ion mobility separation capabilities. The data obtained revealed global differences in cellular protein content between various chaperone prefoldin mutants as well as differential expression of a set of proteins promising to be interesting targets for further investigations.
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Haddad, Iman. "Caractérisation protéomique du matrisome des maladies des petits vaisseaux cérébraux par spectrométrie de masse." Thesis, Université Paris sciences et lettres, 2020. http://www.theses.fr/2020UPSLS026.
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Les maladies des petits vaisseaux cérébraux sont responsables de lésions de la substance blanche du cerveau et d'infarctus cérébrauxprofonds multiples. C'est un ensemble de processus pathologiques, qui affectent les petites artères, les artérioles, veinules oucapillaires cérébraux de moins de 400µm. Le matrisome cérébrovasculaire semble être une voie pathologique convergente entre lesdifférentes maladies des petits vaisseaux de type génétique et de type sporadique. La diversité structurale et physico-chimique desprotéines du matrisome rend cependant leur analyse particulièrement délicate. En effet, certaines protéines du core matrisome,protéines de haut poids moléculaire, sont particulièrement insolubles et d’autres protéines du matrisome associé sont généralementplus petites et moins abondantes. Dans le cadre de cette thèse nous nous proposons de caractériser de manière quantitative etqualitative le matrisome microvasculaire dans les maladies des petits de vaisseaux, ainsi que d'identifier des anomalies communes ouspécifiques à chaque maladie. Pour cela nous avons, dans un premier temps, développé une approche protéomique quantitative enspectrométrie de masse de type label-free sur des vaisseaux cérébraux et périphériques isolés, et nous l’avons validée dans unepremière étude sur un modèlemurin préclinique de CADASIL. Ensuite nous avons appliqué cette approche sur deux autres modèlesmurins pour les maladies génétiques CARASIL et la maladie du collagène de type IV, et deux modèles murins pour l'hypertensionetl'âge, modèle pour leur caractère sporadique. Nous avons développé et validé une nouvelle méthode robuste et sensible pourl’analyse quantitative sans marquage des changements du matrisome des artères cérébrales de souris. En effet, nous avons mis enévidence une protéine commune à toutes ces formes de cSVDs, PRSS23, une serine protéase dans les 5 modèles étudiée et HTRA1,une autre serine protéase, dans CADASIL, la maladie du collagène de type IV et l’âgeDiseases of the small vessels of the brain are responsible for damage to the white matter of the brain and multiple deep braininfarctions. They are the cause of more than 25% of strokes and are the second cause of dementia after Alzheimer's dementia. It is aset of pathological processes, which affect small arteries, arterioles, cerebral venule or capillary of less than 400µm. Thecerebrovascular matrisome seems to be a converging pathological pathway between the various diseases of the small vessels of thegenetic type and of the sporadic type. The matrisome is the set of proteins constituting the extracellular matrix (ECM) as well as theassociated proteins, their roles consist not only in the support and the anchoring of the cells but also in various fundamental processessuch as differentiation, proliferation, Survival or migration of cells. The structural and physico-chemical diversity of these proteins,however, makes their analysis particularly delicate. Within the framework of this thesis we propose to characterize in a quantitativeand qualitative way the microvascular matrisome in the diseases of the small vessels, as well as to identify commonabnormalities orspecific to each disease. For this we have developed a label-free quantitative proteomic approach on cerebral and peripheral vesselsisolated from three preclinical genetic murine models and two murine models for the sporadic character. We have developed andvalidated a new robust and sensitive method for the quantitative non-labeling analysis of changes in the matrisome of mouse cerebralarteries and the application of our method on the arteries of the different mouse models studied has allowed us to identify someavenues. Interesting for each disease independently but also highlighted some common signatures between the different studies
Book chapters on the topic "Label-free quantitative proteomic"
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Liu, Boyan, Danisha Johal, Mitra Serajazari, and Jennifer Geddes-McAlister. "Label-Free Quantitative Proteomic Profiling of Fusarium Head Blight in Wheat." In Methods in Molecular Biology, 287–97. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2124-0_20.
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Wang, Yan, Zhuang Lu, and Lei Wang. "Uncover the Nuclear Proteomic Landscape with Enriched Nuclei Followed by Label-Free Quantitative." In Methods in Molecular Biology, 115–24. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1370-2_12.
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Léger, Thibaut, Camille Garcia, Mathieu Videlier, and Jean-Michel Camadro. "Label-Free Quantitative Proteomics in Yeast." In Methods in Molecular Biology, 289–307. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3079-1_16.
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Moulder, Robert, Young Ah Goo, and David R. Goodlett. "Label-Free Quantitation for Clinical Proteomics." In Methods in Molecular Biology, 65–76. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3524-6_4.
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Levin, Yishai. "CHAPTER 8. Label-free Quantification of Proteins Using Data-Independent Acquisition." In Quantitative Proteomics, 175–84. Cambridge: Royal Society of Chemistry, 2014. http://dx.doi.org/10.1039/9781782626985-00175.
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Soderblom, Erik J., J. Will Thompson, and M. Arthur Moseley. "CHAPTER 6. Overview and Implementation of Mass Spectrometry-Based Label-Free Quantitative Proteomics." In Quantitative Proteomics, 129–53. Cambridge: Royal Society of Chemistry, 2014. http://dx.doi.org/10.1039/9781782626985-00129.
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Schilling, Birgit, Brendan X. MacLean, Alexandria D'Souza, Matthew J. Rardin, Nicholas J. Shulman, Michael J. MacCoss, and Bradford W. Gibson. "CHAPTER 7. MS1 Label-free Quantification Using Ion Intensity Chromatograms in Skyline (Research and Clinical Applications)." In Quantitative Proteomics, 154–74. Cambridge: Royal Society of Chemistry, 2014. http://dx.doi.org/10.1039/9781782626985-00154.
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Haqqani, Arsalan S., John F. Kelly, and Danica B. Stanimirovic. "Quantitative Protein Profiling by Mass Spectrometry Using Label-Free Proteomics." In Methods in Molecular Biology, 241–56. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-188-8_17.
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Neilson, Karlie A., Tim Keighley, Dana Pascovici, Brett Cooke, and Paul A. Haynes. "Label-Free Quantitative Shotgun Proteomics Using Normalized Spectral Abundance Factors." In Methods in Molecular Biology, 205–22. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-360-2_17.
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Wieczorek, Samuel, Florence Combes, Hélène Borges, and Thomas Burger. "Protein-Level Statistical Analysis of Quantitative Label-Free Proteomics Data with ProStaR." In Methods in Molecular Biology, 225–46. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9164-8_15.
Full textConference papers on the topic "Label-free quantitative proteomic"
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Singh, Chandra K., Satwinderjeet Kaur, Jasmine George, Molly C. Pellitteri-Hahn, Cameron O. Scarlett, and Nihal Ahmad. "Abstract 4533: Mechanism of anti-proliferative effects of sanguinarine in pancreatic cancer cells: A label-free quantitative proteomics approach." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-4533.
Full textReports on the topic "Label-free quantitative proteomic"
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Chen, Xiaole, Peng Wang, Yunquan Luo, Yi-Yu Lu, Wenjun Zhou, Mengdie Yang, Jian Chen, Zhi-Qiang Meng, and Shi-Bing Su. Therapeutic Efficacy Evaluation and Underlying Mechanisms Prediction of Jianpi Liqi Decoction for Hepatocellular Carcinoma. Science Repository, September 2021. http://dx.doi.org/10.31487/j.jso.2021.02.04.sup.
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Objective: The aim of this study was to assess the therapeutic effects of Jianpi Liqi decoction (JPLQD) in hepatocellular carcinoma (HCC) and explore its underlying mechanisms. Methods: The characteristics and outcomes of HCC patients with intermediate stage B who underwent sequential conventional transcatheter arterial chemoembolization (cTACE) and radiofrequency ablation (RFA) only or in conjunction with JPLQD were analysed retrospectively. The plasma proteins were screened using label-free quantitative proteomics analysis. The effective mechanisms of JPLQD were predicted through network pharmacology approach and partially verified by ELISA. Results: Clinical research demonstrated that the Karnofsky Performance Status (KPS), traditional Chinese medicine (TCM) syndrome scores, neutropenia and bilirubin, median progression-free survival (PFS), and median overall survival (OS) in HCC patients treated with JPLQD were superior to those in patients not treated with JPLQD (all P<0.05). The analysis of network pharmacology, combined with proteomics, suggested that 52 compounds targeted 80 potential targets, which were involved in the regulation of multiple signaling pathways, especially affecting the apoptosis-related pathways including TNF, p53, PI3K-AKT, and MAPK. Plasma IGFBP3 and CA2 were significantly up-regulated in HCC patients with sequential cTACE and RFA therapy treated with JPLQD than those in patients not treated with JPLQD (P<0.001). The AUC of the IGFBP3 and CA2 panel, estimated using ROC analysis for JPLQD efficacy evaluation, was 0.867. Conclusion: These data suggested that JPLQD improves the quality of life, prolongs the overall survival, protects liver function in HCC patients, and exhibits an anticancer activity against HCC. IGFBP3 and CA2 panels may be potential therapeutic targets and indicators in the efficacy evaluation for JPLQD treatment, and the effective mechanisms involved in the regulation of multiple signaling pathways, possibly affected the regulation of apoptosis.