Academic literature on the topic 'Integrated proteomic analysi'
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Journal articles on the topic "Integrated proteomic analysi"
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Senavirathna, Lakmini, Cheng Ma, Ru Chen, and Sheng Pan. "Spectral Library-Based Single-Cell Proteomics Resolves Cellular Heterogeneity." Cells 11, no. 15 (August 7, 2022): 2450. http://dx.doi.org/10.3390/cells11152450.
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Dissecting the proteome of cell types and states at single-cell resolution, while being highly challenging, has significant implications in basic science and biomedicine. Mass spectrometry (MS)-based single-cell proteomics represents an emerging technology for system-wide, unbiased profiling of proteins in single cells. However, significant challenges remain in analyzing an extremely small amount of proteins collected from a single cell, as a proteome-wide amplification of proteins is not currently feasible. Here, we report an integrated spectral library-based single-cell proteomics (SLB-SCP) platform that is ultrasensitive and well suited for a large-scale analysis. To overcome the low MS/MS signal intensity intrinsically associated with a single-cell analysis, this approach takes an alternative approach by extracting a breadth of information that specifically defines the physicochemical characteristics of a peptide from MS1 spectra, including monoisotopic mass, isotopic distribution, and retention time (hydrophobicity), and uses a spectral library for proteomic identification. This conceptually unique MS platform, coupled with the DIRECT sample preparation method, enabled identification of more than 2000 proteins in a single cell to distinguish different proteome landscapes associated with cellular types and heterogeneity. We characterized individual normal and cancerous pancreatic ductal cells (HPDE and PANC-1, respectively) and demonstrated the substantial difference in the proteomes between HPDE and PANC-1 at the single-cell level. A significant upregulation of multiple protein networks in cancer hallmarks was identified in the PANC-1 cells, functionally discriminating the PANC-1 cells from the HPDE cells. This integrated platform can be built on high-resolution MS and widely accepted proteomic software, making it possible for community-wide applications.
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Han, Mee-Jung, and Sang Yup Lee. "The Escherichia coli Proteome: Past, Present, and Future Prospects." Microbiology and Molecular Biology Reviews 70, no. 2 (June 2006): 362–439. http://dx.doi.org/10.1128/mmbr.00036-05.
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SUMMARY Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects.
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Bhawal, Ruchika, Ann L. Oberg, Sheng Zhang, and Manish Kohli. "Challenges and Opportunities in Clinical Applications of Blood-Based Proteomics in Cancer." Cancers 12, no. 9 (August 27, 2020): 2428. http://dx.doi.org/10.3390/cancers12092428.
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Blood is a readily accessible biofluid containing a plethora of important proteins, nucleic acids, and metabolites that can be used as clinical diagnostic tools in diseases, including cancer. Like the on-going efforts for cancer biomarker discovery using the liquid biopsy detection of circulating cell-free and cell-based tumor nucleic acids, the circulatory proteome has been underexplored for clinical cancer biomarker applications. A comprehensive proteome analysis of human serum/plasma with high-quality data and compelling interpretation can potentially provide opportunities for understanding disease mechanisms, although several challenges will have to be met. Serum/plasma proteome biomarkers are present in very low abundance, and there is high complexity involved due to the heterogeneity of cancers, for which there is a compelling need to develop sensitive and specific proteomic technologies and analytical platforms. To date, liquid chromatography mass spectrometry (LC-MS)-based quantitative proteomics has been a dominant analytical workflow to discover new potential cancer biomarkers in serum/plasma. This review will summarize the opportunities of serum proteomics for clinical applications; the challenges in the discovery of novel biomarkers in serum/plasma; and current proteomic strategies in cancer research for the application of serum/plasma proteomics for clinical prognostic, predictive, and diagnostic applications, as well as for monitoring minimal residual disease after treatments. We will highlight some of the recent advances in MS-based proteomics technologies with appropriate sample collection, processing uniformity, study design, and data analysis, focusing on how these integrated workflows can identify novel potential cancer biomarkers for clinical applications.
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Sobolev, Vladimir V., Anna G. Soboleva, Elena V. Denisova, Eva A. Pechatnikova, Eugenia Dvoryankova, Irina M. Korsunskaya, and Alexandre Mezentsev. "Proteomic Studies of Psoriasis." Biomedicines 10, no. 3 (March 7, 2022): 619. http://dx.doi.org/10.3390/biomedicines10030619.
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In this review paper, we discuss the contribution of proteomic studies to the discovery of disease-specific biomarkers to monitor the disease and evaluate available treatment options for psoriasis. Psoriasis is one of the most prevalent skin disorders driven by a Th17-specific immune response. Although potential patients have a genetic predisposition to psoriasis, the etiology of the disease remains unknown. During the last two decades, proteomics became deeply integrated with psoriatic research. The data obtained in proteomic studies facilitated the discovery of novel mechanisms and the verification of many experimental hypotheses of the disease pathogenesis. The detailed data analysis revealed multiple differentially expressed proteins and significant changes in proteome associated with the disease and drug efficacy. In this respect, there is a need for proteomic studies to characterize the role of the disease-specific biomarkers in the pathogenesis of psoriasis, develop clinical applications to choose the most efficient treatment options and monitor the therapeutic response.
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Petralia, Francesca, Nicole Tignor, Dmitri Rykunov, Boris Revas, Shrabanti Chowdhury, Azra Krek, Pichae Raman, et al. "TBIO-19. INTEGRATED GENOMIC, PROTEOMIC AND PHOSPHOPROTEOMIC ANALYSIS OF SEVEN TYPES OF PEDIATRIC BRAIN CANCER." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii470. http://dx.doi.org/10.1093/neuonc/noaa222.846.
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Abstract We performed a comprehensive proteogenomic analysis across seven childhood brain tumors for a deeper understanding of their functional biology. Whole genome sequencing, RNAseq, quantitative proteomic profiling and phosphoproteomics were performed on 219 fresh frozen tumor samples representing the histologic diagnoses of: low grade astrocytoma (93), ependymoma (32), high grade astrocytoma (26), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16) and atypical teratoid rhabdoid tumor (12). Unsupervised clustering analysis based on proteomics data reveals eight clusters with distinct protein profiles and pathway activities. While some clusters coincide with histologic diagnoses, a couple of clusters appear to be a mixture of different diagnoses, including one cluster consisting of “aggressive” tumors characterized by poor survival and high stemness scores. By integrating proteomic data with RNAseq and WGS data, we characterize the impact of mutations (H3K27M, BRAFV600E, BRAF fusion) and CNVs upon the proteome across various diagnoses. Multiomics based kinase-substrate association analysis and co-expression network analysis reveal targetable active kinase networks within these tumors. Proteomic data reveals unique biology associated with H3K27M mutation status in HGG and BRAF aberrations in LGG. Characterization of the tumor microenvironment through deconvolution analyses based on multi-omics data reveals 5 distinct tumor clusters associated with different populations of infiltrating immune cells and the relative activity of the immune system based upon the expression of pro-inflammation or immunosuppressive markers. This study reports the first large-scale deep comprehensive proteogenomic analysis crossing traditional histologic boundaries to uncover foundational pediatric brain tumor biology including functional insight that helps drive translational efforts.
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Wu, Jingyu, Zhifang Hao, Chen Ma, Pengfei Li, Liuyi Dang, and Shisheng Sun. "Comparative proteogenomics profiling of non-small and small lung carcinoma cell lines using mass spectrometry." PeerJ 8 (April 23, 2020): e8779. http://dx.doi.org/10.7717/peerj.8779.
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Background Evidences indicated that non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) might originate from the same cell type, which however ended up to be two different subtypes of lung carcinoma, requiring different therapeutic regimens. We aimed to identify the differences between these two subtypes of lung cancer by using integrated proteome and genome approaches. Methods and Materials Two representative cell lines for each lung cancer subtype were comparatively analysed by quantitative proteomics, and their corresponding transcriptomics data were obtained from the Gene Expression Omnibus database. The integrated analyses of proteogenomic data were performed to determine key differentially expressed proteins that were positively correlated between proteomic and transcriptomic data. Result The proteomics analysis revealed 147 differentially expressed proteins between SCLC and NSCLC from a total of 3,970 identified proteins. Combined with available transcriptomics data, we further confirmed 14 differentially expressed proteins including six known and eight new lung cancer related proteins that were positively correlated with their transcriptomics data. These proteins are mainly involved in cell migration, proliferation, and invasion. Conclusion The proteogenomic data on both NSCLC and SCLC cell lines presented in this manuscript is complementary to existing genomic and proteomic data related to lung cancers and will be crucial for a systems biology-level understanding of the molecular mechanism of lung cancers. The raw mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015270.
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Wang, Xuchu. "Protein and Proteome Atlas for Plants under Stresses: New Highlights and Ways for Integrated Omics in Post-Genomics Era." International Journal of Molecular Sciences 20, no. 20 (October 21, 2019): 5222. http://dx.doi.org/10.3390/ijms20205222.
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In the post-genomics era, integrative omics studies for biochemical, physiological, and molecular changes of plants in response to stress conditions play more crucial roles. Among them, atlas analysis of plants under different abiotic stresses, including salinity, drought, and toxic conditions, has become more important for uncovering the potential key genes and proteins in different plant tissues. High-quality genomic data and integrated analyses of transcriptomic, proteomic, metabolomics, and phenomic patterns provide a deeper understanding of how plants grow and survive under environmental stresses. This editorial mini-review aims to synthesize the 27 papers including two timely reviews that have contributed to this Special Issue, which focuses on concluding the recent progress in the Protein and Proteome Atlas in plants under different stresses. It covers various aspects of plant proteins ranging from agricultural proteomics, structure and function of proteins, novel techniques and approaches for gene and protein identification, protein quantification, proteomics for post-translational modifications (PTMs), and new insights into proteomics. The proteomics-based results in this issue will help the readers to gain novel insights for the understanding of complicated physiological processes in crops and other important plants in response to stressed conditions. Furthermore, these target genes and proteins that are important candidates for further functional validation in economic plants and crops can be studied.
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Vowinckel, Jakob, Thomas Corwin, Jonathan Woodsmith, Tobias Treiber, Roland Bruderer, Lukas Reiter, Eike-Christin von Leitner, Karel Novy, Hartmut Juhl, and Oliver Rinner. "Proteome and phospho-proteome profiling for deeper phenotype characterization of colorectal cancer heterogeneity." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e15536-e15536. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e15536.
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e15536 Background: The rise of precision oncology therapeutics requires deep understanding of the molecular mechanisms implicated in cancer biology. Colorectal cancer (CRC) is one of the first solid tumors to be molecularly characterized by defined genes and pathways. Advances in tumor profiling have revealed a profound molecular heterogeneity in CRC leading to the definition of several consensus molecular subtypes (CMS). However, this molecular heterogeneity is still largely defined on the genomic and transcriptomics level. To complement the understanding of genetically defined molecular subgroups, we performed large-scale deep proteomic and phospho-proteomic profiling of CRC patient biopsies and adjacent healthy control tissue, which has enabled to explore the phenotype and obtain more functional insights in cancer biology. Methods: Sample processing from 5-10 mg of tissue per sample was performed using a liquid handling robot. Phospho-peptide enrichment was carried out with a Kingfisher Flex device and MagReSyn Ti-IMAC magnetic beads. Data-Independent Acquisition (DIA) LC-MS/MS was performed on multiple platforms consisting of a Thermo Scientific Q Exactive HF-X mass spectrometer coupled to a Waters M-Class LC. Chromatography was operating at 5 µL/min, and separation was achieved using 45 min (whole proteome) and 60 min (phospho-proteome) gradients. Results: Indivumed has built IndivuType, the world’s first multi-omics database for individualized cancer therapy, analyzing the highest quality cancer biospecimens to generate the most comprehensive dataset, including genomics, transcriptomics, proteomics, and clinical outcome information. Enabled by the DIA technology, a mass spectrometric method developed by Biognosys that obtains peptide fragmentation data in a highly parallelized way with high sensitivity, more than 7,000 proteins in the whole proteome and 20,000 phospho-peptides in the phospho-proteome workflow were profiled across more than 900 resected tissue samples of various CMS of CRC. The resulting proteome and phospho-proteome data were integrated into the IndivuType database and cross-analyzed with genomic and transcriptomic markers. Through this combined analysis, novel insights in clinically relevant signaling pathways in CRC subtypes were revealed. Conclusions: The deep phenotypic profiling of cancer samples, using next generation proteomics and phospho-proteomics, has enabled us to go beyond the genomic level in the characterization of tumor molecular heterogeneity. This multi-omics approach provides a solid foundation to advance the understanding of cancer biology, unravel key molecular events, and support the identification of novel therapeutic targets for precision medicine in CRC.
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Carvalho, Paulo C., Diogo B. Lima, Felipe V. Leprevost, Marlon D. M. Santos, Juliana S. G. Fischer, Priscila F. Aquino, James J. Moresco, John R. Yates, and Valmir C. Barbosa. "Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0." Nature Protocols 11, no. 1 (December 10, 2015): 102–17. http://dx.doi.org/10.1038/nprot.2015.133.
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Pruess, Manuela, Paul Kersey, and Rolf Apweiler. "Integrating Genomic and Proteomic Data: The Integr8 Project." Journal of Integrative Bioinformatics 1, no. 1 (December 1, 2004): 108–15. http://dx.doi.org/10.1515/jib-2004-9.
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Summary Integr8 (http://www.ebi.ac.uk/integr8/) has been developed to provide an integration layer for the exploitation of genomic and proteomic data. High-quality databases from major bioinformatics centres in Europe are included, and some core data and the relationships of biological entities to each other and to entries in other databases are stored. Thus, a framework exists that allows for new kinds of data to be integrated, and an entity-centric view of complete genomes and proteomes is offered. Integr8 is an automatically populated database, providing different entry points to the data, depending on the user’s entity of interest. The Proteome Analysis database for statistical analysis and the Genome Reviews for annotated genome information are the main developments within the Integr8 project. With the BioMart application, an interactive querying tool for performing customisable proteome analysis and data mining is offered. Future developments will especially focus on the Genome Reviews, including mapping not yet annotated protein sequences onto their corresponding genomes, generating new predictions for non-coding RNA genes, and generally extending the scope to lower metazoan organisms.
Dissertations / Theses on the topic "Integrated proteomic analysi"
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Caterino, Cinzia. "The aging synapse: an integrated proteomic and transcriptomic analysis." Doctoral thesis, Scuola Normale Superiore, 2019. http://hdl.handle.net/11384/86004.
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An important hallmark of aging is the loss of proteostasis, which can lead to the formation of protein
aggregates and mitochondrial dysfunction in neurons. Although it is well known that protein
synthesis is finely regulated in the brain, especially at synapses, where mRNAs are locally translated in
activity-dependent manner, little is known as to the changes in the synaptic proteome and
transcriptome during aging. Therefore, this work aims to elucidate the relationship between
transcriptome and proteome at soma and synaptic level during aging.
Cerebral cortices were isolated from 3 weeks-old mice, 5 months-old and 18 months-old mice and
synaptosomal fraction was extracted by ultracentrifugation on discontinuous sucrose gradient. The
fraction was then analyzed by Data Independent Analysis (DIA) Mass Spectrometry and the resulting
data were analyzed using Spectronaut software. RNA was also extracted and analyzed by ribo-zero
RNA-seq. Data were analyzed and combined with R software.
Proteomic and transcriptomic data analysis revealed that, in young animals, proteins and transcripts
are correlated and synaptic regulation is driven by changes in the soma. During aging, there is a
decoupling between transcripts and proteins and between somatic and synaptic compartments. For
example, there is an increase of ribosomal proteins at synapses that is not mirrored by a concomitant
increase at somatic level. Furthermore, soma-synapse gradient of ribosomal genes changes upon
aging, i.e. ribosomal transcripts are less abundant and ribosomal proteins are more abundant in
synaptic compartment of old mice with respect to younglings.
Mass spectrometry analysis of synaptic protein aggregates revealed that they are particularly rich in
ribosomal proteins and also of some components of lysosomes and proteasome, suggesting that loss
of proteostasis and inefficient degradation leads to aggregation of ribosomes in synaptic
compartment. Strikingly, Desmoplakin, a structural constituent of desmosomes, was also highly
abundant in synaptic aggregates. This study suggests that aging affects both the local translational
machinery and the trafficking of transcripts and proteins.
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Parkinson, Erika. "An integrated proteomic and bioinformatic analysis for the diagnosis and prognosis of cancer." Thesis, Nottingham Trent University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.442148.
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The advent of proteomics and high-throughput technologies has allowed scientists to derive protein expression patterns of potential use in predictive medicine. The application of bioinformatics to analyse complex data makes it possible to identify important protein biomarkers. These biomarkers may have predictive capability to determine, for example, the presence and progression of disease and how an individual patient might respond to therapy. Mass spectrometry (MS) has increasingly become the method of choice for the analysis of complex samples and new MS systems have been developed that can rapidly profile and generate proteomic 'fingerprints' from tissue and body fluids. In particular, MALDI mass spectrometry coupled with Ciphergen® chip technology (SELDI MS) has been widely used to identify discriminatory patterns to distinguish patients at different clinical stages of disease, for example, in ovarian, prostate, colon and breast cancer. All of these studies incorporate the use of computer algorithms to mine the proteomic data obtained from the mass spectra, allowing large cohorts of samples to be included into the analysis. The aim of this study was to introduce the use of MS and bioinfonnatics to analyse the cancer proteome, in particular melanoma and breast cancer and to investigate the information obtained from profiling cell lines, tissue and serum samples, as well as evaluating the type of analytical methods currently available. The methods used in this study for sample preparation and analysis demonstrate that good quality proteomic data from cell lines, tissue and serum can be obtained and that it is possible to generate discriminatory protein profiles that correlate with clinical outcomes when analysed using Artificial Neural Networks (ANNs). Through the analysis of the proteome of melanoma cell lines, it is possible to classify samples according to the presence of specific genetic mutations, the site of the tumour sample from which the cell line was derived, as well as the overall survival of a patient. Comparison of melanoma cell line proteomes and their tumour tissue of origin revealed that both sample types were able to provide discriminating patterns that correlated to clinical outcomes. This finding has significance for future proteomic-based biomarker discovery research where it is possible to use cell lines in place of "precious" tumour tissue for the identification of clinically relevant biomarkers. The presence of a basal phenotype, which signifies the aggressive nature of breast cancer, can be identified from the proteomic profiling of patients' breast cancer tissue. The analysis of melanoma patient serum was investigated and patterns that predicted the stage of disease, as well as disease progression, were identified, using SELDI MS and ANNs. These results demonstrate that it is possible to obtain clinically valid information from the proteome of samples derived from melanoma and breast cancer patients through the use of SELDI MS and ANN analysis. Although SELDI MS has proven useful in generating protein profiles that can be used for identifying patients with different clinical outcomes, this technology has limitations. One aspect of the study was to determine if similar, or more accurate, discriminatory analysis could be achieved using higher resolution and higher sensitivity MALDI instrumentation. A set of melanoma cell line samples were subjected to SELDI MS and MALDI MS analysis and the data from both methods were analysed in the same way by ANNs. Slightly different sample preparation methods were used prior to MS analysis, thus the spectra obtained by SELDI MS and MALDI MS was dissimilar; the data revealed that MALDI MS did not improve upon the accuracy of classifying samples. The work presented demonstrates a proof-of-principle of the different types of information that can be obtained from samples derived from melanoma and breast cancer patients. It has also been revealed that the analysis of MS spectra by ANNs can be used for predicting blind datasets which is not necessarily dependent on the MS method used; however, this is likely to have significant implications for biomarker identification as the different methods used will reveal different disease-associated proteins.
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Mueller, Michael. "Integrated analysis of proteomics data to assess and improve the scope of mass spectrometry based genome annotation." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611790.
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Szapiel, Nicolas. "Glc7-E101Q is a novel tool for integrated genomic and proteomic analysis of PP1Glc7 phosphatase functional networks in Saccharomyces cerevisiae." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101656.
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Reversible phosphorylation is a major mechanism for regulating the activity, localization and stability of proteins required for vital cellular processes such as glucose metabolism, gene expression, establishment of polarity, mitosis and cytokinesis. Phospho-regulation is driven by the activities of kinases and phosphatases. Together, these enzymes account for ∼3% of eukaryotic genomes and it is estimated that 30% of the eukaryotic proteome is composed of phospho-proteins. Protein kinases (PKs) have been studied extensively, however relatively little is known regarding the signaling networks of protein phosphatases (PPases). The identification of PPase functional networks has been slow due to the redundant nature of the majority of PPases, the complexity of their substrate recognition in vivo, and the lack of large-scale analyses that would facilitate network analysis. We hypothesized that large-scale analysis of genetic interactions using the Synthetic Genetic Array (SGA) and proteomic analyses using 2D-PAGE Difference Gel Electrophoresis (DiGE) could reveal PPase functional networks. Here, we apply this approach to the essential and conserved PP1 PPase Glc7 as it regulates numerous cellular processes in budding yeast. For this study, we created a glc7 hypomorphic mutant (glc7-E101Q) suited for both SGA and DiGE analyses. SGA analysis of glc7-E101Q revealed a broad network of 147 synthetic sick/lethal (SSL) and 178 synthetic rescue (SR) interactions. DiGE comparison of the glc7-E101Q proteome relative to wild-type at medium-resolution (∼1000 proteins) revealed alterations in 39 proteins that changed as a consequence of both the mutation and growth conditions. One of the proteins identified in this analysis was Eno1, a non-essential enolase that is mis-regulated in the presence of glucose and identified a SR mutation in the glc7-E101Q SGA. Subsequent phenotypic analysis suggests a novel, non-metabolic role for Eno1 in the Glc7 interaction network. Our results reveal that parallel analysis, using SGA and DIGE, can reveal novel functions and networks that a single analysis may not detect.
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MATTE', Alessandro. "Integrated proteomic analysis of normal and diseased red cells. Role of physiological stresses in erythrocyte signaling pathways." Doctoral thesis, 2009. http://hdl.handle.net/11562/337506.
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Il globulo rosso è una cellula altamente specializzata per il trasporto di ossigeno e anidride
carbonica. Studi di fisiopatologia eritroide hanno permesso di riconoscere 4 compartimenti
fondamentali: la membrana, il citoscheletro, gli enzimi citosolici e l’emoglobina. Alterazioni
a carico di uno di questi compartimenti sono responsabili di profonde anomalie
nell’organizzazione funzionale degli eritrociti, che si traducono clinicamente in anemie
ereditarie o acquisite.
Le anemie ereditarie sono sostenute da mutazioni monogeniche e la loro diffusione e’
legata alla selezione positiva indotta dalla malaria. Tra i disordini ereditari dei globuli rossi,
l’organizzazione mondiale della sanita’ (OMS) ha identificato le emoglobinopatie e in
particolare le β-talassemie come malattie emergenti in tutte le aree geografiche del
mondo.
L’enorme differenza tra il genotipo e il fenotipo clinico dell’emoglobinopatie suggerisce che
eventi post-traslazionali possano essere giocare un ruolo importante nelle interazioni
proteina-proteina coinvolte nelle diverse funzioni cellulari, influenzando in ultima analisi le
manifestazioni cliniche di queste malattie.
Tra le modificazioni post-traslazionali a carico delle proteine gli eventi di
fosforilazione/defosforilazione sostenuti da chinasi/fosfatasi costituiscono uno degli
elementi chiave nella trasduzione del segnale coinvolti nell’omeostasi cellulare.
Gli scopi del presente progetto di ricerca sono stati quelli di caratterizzare vie di segnale
intracellulare in risposta a stress in globuli rossi normali e patologici.
Nella prima parte della ricerca abbiamo analizzato l’espressione e la localizzazione di
perossiredossina-2 (Prx2) in globuli rossi di 2 modelli murini di β-talassemia caratterizzati
da diversa gravita’ del fenotipo ematologico (Hbbth/th e Hbbth3/+). Nei globuli rossi β-
talassemici abbiamo descritto (paper submitted to Blood, 2009) un’aumentata espressione
di Prx2 ma una riduzione nella quantità di Prx2 legata alla membrana a fronte di un grave
danno ossidativo. Utilizzando un’analisi di proteomica integrata abbiamo dimostrato che
una parte di Prx2 legata alla membrana e’ fosforilata e che questa modificazione posttraslazionale
influenza la funzione di Prx2 e la sua fisiologica risposta al danno ossidativo.
Nella seconda parte del progetto sono stati analizzati gli effetti della deossigenazione sul
proteoma di membrana dei globuli rossi di topi geneticamente deficienti di Fgr, Hck e Lyn,
chinasi della famiglia del Src (fgr-/-hck-/-; fgr-/-hck-/-lyn-/-). Sulla base di studi preliminari
in proteomica classica, abbiamo condotto un’analisi comparativa del fosfoproteoma della
membrana dei globuli rossi dei 3 ceppi murini esposti a deossigenazione. Questi dati ci
hanno permesso di individuare il nuovo ed importante ruolo di chinasi della famiglia del
Src e di Syk nella trasduzione del segnale in risposta all’ipossia nei globuli rossi.
Lo sviluppo di queste metodologie e l’acquisizione di dati scientifici originali su nuove vie
di trasduzione del segnale in globuli rossi normali e patologici ha permesso di contribuire
allo studio della modulazione delle heat shock protein (HSP27; HSP70) e della Prx2 in
globuli rossi di pazienti affetti da anemia falciforme (Proteomics Clin. Appl. 2008, 2, 706-
719) e allo studio della modulazione degli eventi di fosforilazione della banda 3 da parte di
Syk in globuli rossi normali e di pazienti affetti da deficit di Glucosio-6-fosfato-deidrogenasi
(Biochem J. 2009, 418, 359-367).Peroxiredoxin 2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells (RBCs) and is involved at least in part in defence against oxidative stress. Here, we examined the expression and localization of Prx2 in RBCs from two murine models of β thalassemia (Hbbth3/+, Hbbth/th) and in normal RBCs treated with phenylhydrazine (PHZ) as β thalassemic-like RBCs. Prx2 expression was higher in β thalassemic mouse models than in normal RBCs without effects of cell density/age. Dimeric Prx2 was only detectable in β thalassemic RBC lysates and related with the severity of the hematological phenotype. Although β thalassemic RBC membrane is characterized by a severe oxidative damage, we found Prx2 translocated from the membrane to the cytosol, without detectable dimers as also observed in β thalassemic-like RBCs. We generated 2D maps of the phosphoenriched RBC membrane proteins, and in the area where Prx2 was expected to migrate, anti-phosphotyrosine staining was observed, which was further identified as Prx2 by both mass-spectrometric and immunoblot-analysis. These data demonstrate that a population of Prx2 associated with the membrane is tyrosine-phosphorylated and that tyrosine phosphorylation might regulate both the oligomeric state and membrane association of Prx2, regardless of the oxidation state of the cell.
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TOMELLERI, Carlo. "Integrated analysis of novel signal transduction pathways in red cells from patients with neuroacanthocytosis." Doctoral thesis, 2012. http://hdl.handle.net/11562/395336.
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Il termine neuroacantocitosi (NA) raggruppa diverse malattie genetiche rare che condividono manifestazioni neurologiche simili e la presenza di globuli rossi stellati nella circolazione periferica, gli acantociti. Le due principali malattie classificate come NA sono Corea acantocitosi (ChAc) e sindrome di McLeod (MLS).
Poiché la presenza di acantociti è una caratteristica comune di questi disordini, lo studio dei meccanismi alla base della loro formazione può aiutare a comprendere la patogenesi delle NA. In questa tesi viene presentata una serie di studi sui meccanismi di signaling e sulle modificazioni strutturali di globuli rossi di pazienti di ChAc e MLS.
Nel primo studio abbiamo analizzato con tecniche di proteomica la fosforilazione in tirosina di globuli rossi di pazienti affetti da ChAc .
Nei globuli rossi di questi pazienti abbiamo riscontrato un aumento della fosforilazione in tirosina su diverse proteine di membrana e legate alla membrana tra cui banda 3, β-spectrina e adducina. In particolare, la fosforilazione sul residuo Tyr-904 della banda 3, target della chinasi Lyn, era molto elevata, mentre sul residuo Tyr-8 della stessa proteina, target della chinasi Syk, non abbiamo riscontrato un aumento della fosforilazione. Nei pazienti di ChAc, la fosforilazione della banda 3 da parte di Lyn è indipendente dal meccanismo canonico di fosforilazione sequenziale mediato da Syk.
Le alterazioni dell’organizzazione delle proteine di membrana correlate con ChAc sembrano quindi essere il risultato di un aumento della fosforilazione in tirosina che porta a cambiamenti nel legame della banda 3 con i ponti multiproteici tra la membrana e il citoscheletro. Proponiamo quindi quest’alterazione nell’associazione tra membrana e citoscheletro mediata da fosforilazione in tirosina come un nuovo meccanismo che porta alla formazione di acantociti in ChAc.
Nel secondo studio abbiamo combinato i nostri set di dati fosfo-proteomici su eritrociti di pazienti di ChAc e MLS con l’analisi topologica di network proteici per predirre quali sub-network della trasduzione del segnale possano essere coinvolti nella formazione degli acantociti.
Abbiamo identificato tutte le interazioni che legano le due proteine mutate nelle due patologie in esame (rispettivamente coreina e XK) con le proteine differenzialmente fosforilate dei nostri dati sperimentali. Quindi, abbiamo analizzato nello specifico solo cluster di proteine coinvolte nella trasduzione del segnale che interagiscono molto strettamente tra loro e che possono essere coinvolte nella formazione di acantociti in entrambe le patologie. Abbiamo identificato un cluster di 14 chinasi che possono essere coinvolte in tale processo e meritano ulteriori approfondimenti.
Come studio preliminare nel contesto di una collaborazione internazionale abbiamo analizzato globuli rossi da pazienti affetti da Neurodegenerazione con Accumulo di Ferro nel Cervello (NBIA) e di loro parenti di primo grado. Il nostro scopo era quello di determinare se fossero presenti acantociti nei pazienti e nei soggetti correlati ma privi di sintomi clinici e di studiare le caratteristi che strutturali dei loro eritrociti.
Nell’ultimo studio abbiamo validato con l’applicazione a tecniche di analisi proteomica un nuovo copolimero basato su acrilamide e polivinil alcool modificato con gruppi olefinici. Questo nuovo idrogel è semplice da maneggiare anche a basse concentrazioni e la sua macroporosità lo rende particolarmente adatto alla separazione di proteine ad alto peso molecolare quale la coreina.Neuroacanthocytosis (NA) is a group of rare genetic disorders that share similar neurological clinical manifestations and the presence of thorny red cells in the peripheral circulation, the acanthocytes. The two core NA diseases are Chorea-Acanthocytosis (ChAc) and McLeod Syndrome (MLS). Since acanthocytes are an hallmark of NA, studying the mechanisms underlying the generation of acanthocytes might shed light on the pathogenesis of NA syndromes. Here, we present a set of studies on the signaling mechanisms and structural changes in red cells from ChAc and MLS patients. In the first study, we evaluated tyrosine phosphorylation of red cells from ChAc patients by proteomics analysis. Increased Tyr-phosphorylation state of several membrane proteins including band 3, β-spectrin and adducin was found in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs band 3 Tyr-phosphorylation by Lyn was independent of the canonical Syk mediated pathway. The ChAc-associated alterations in RBC membrane-protein organization appear to be the result of increased Tyr-phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton. We propose this altered association between cytoskeleton and membrane proteins as a novel mechanism in the generation of acanthocytes in ChAc. In the second study, we combined phosphoproteomics datasets on ChAc and MLS with network topology analysis to predict signaling sub-networks involved in acanthocyte generation. We identified all the interactomic shortest paths linking the two proteins mutated in NA syndromes, respectively chorein and XK, to the differentially phosphorylated proteins in our proteomics data. Then, we refined the analysis considering only restricted clusters of highly interacting signaling proteins which can be involved in acanthocyte formation in both diseases. We identified a cluster of 14 kinases that might be related to red cell shape alterations and deserve further investigation. As preliminary study in the context of an international collaboration we analyzed red cells from Neurodegeneration with Brain Iron Accumulation (NBIA) patients and their first degree relatives. Our aim was to assess the presence of acanthocytes in these subjects and to study their structural characteristics. In the last study, we validated a new co-polymer based on acrylamide and polyvinyl alcohol bearing olefinic moieties in proteomic analysis of red cells. This new hydrogel is easy to handle and its macroporosity makes it suitable for the separation of high molecular weight proteins such as chorein.
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Pan, Chongle. "An integrated experimental and computational approach to proteomics scaling from high resolution qualitative analysis to quantitative measurements with confidence evaluation /." 2006. http://etd.utk.edu/2006/PanChongle.pdf.
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Thesis (Ph. D.) -- University of Tennessee, Knoxville, 2006.Title from title page screen (viewed on February 2, 2007). Thesis advisor: Robert L. Hettich. Vita. Includes bibliographical references.
Books on the topic "Integrated proteomic analysi"
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Sklar, Larry A., ed. Flow Cytometry for Biotechnology. Oxford University Press, 2005. http://dx.doi.org/10.1093/oso/9780195183146.001.0001.
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Flow cytometry is a sensitive and quantitative platform for the measurement of particle fluorescence. In flow cytometry, the particles in a sample flow in single file through a focused laser beam at rates of hundreds to thousands of particles per second. During the time each particle is in the laser beam, on the order of ten microseconds, one or more fluorescent dyes associated with that particle are excited. The fluorescence emitted from each particle is collected through a microscope objective, spectrally filtered, and detected with photomultiplier tubes. Flow cytometry is uniquely capable of the precise and quantitative molecular analysis of genomic sequence information, interactions between purified biomolecules and cellular function. Combined with automated sample handling for increased sample throughput, these features make flow cytometry a versatile platform with applications at many stages of drug discovery. Traditionally, the particles studied are cells, especially blood cells; flow cytometry is used extensively in immunology. This volume shows how flow cytometry is integrated into modern biotechnology, dealing with issues of throughput, content, sensitivity, and high throughput informatics with applications in genomics, proteomics and protein-protein interactions, drug discovery, vaccine development, plant and reproductive biology, pharmacology and toxicology, cell-cell interactions and protein engineering.
Book chapters on the topic "Integrated proteomic analysi"
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Osiri, John K., Hamed Shadpour, Małgorzata A. Witek, and Steven A. Soper. "Integrated Multifunctional Microfluidics for Automated Proteome Analyses." In Microfluidics, 261–94. Berlin, Heidelberg: Springer Berlin Heidelberg, 2011. http://dx.doi.org/10.1007/128_2011_152.
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Pettifer, Steve R., James R. Sinnott, and Teresa K. Attwood. "Integrated Approaches for Bioinformatic Data Analysis and Visualization - Challenges, Opportunities and New Solutions." In Data Analysis and Visualization in Genomics and Proteomics, 135–52. Chichester, UK: John Wiley & Sons, Ltd, 2005. http://dx.doi.org/10.1002/0470094419.ch9.
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Wang, Jiangxin, Gang Wu, Lei Chen, and Weiwen Zhang. "Integrated Analysis of Transcriptomic and Proteomic Datasets Reveals Information on Protein Expressivity and Factors Affecting Translational Efficiency." In Methods in Molecular Biology, 123–36. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/7651_2015_242.
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Giri, Archana, and Charu Chandra Giri. "Recent Trends in Elicitation and Secondary Metabolic Pathway Analysis in Medicinal Plants: An Integrated Transcriptomics and Proteomics Approach." In Medicinal and Aromatic Plants of the World, 403–30. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-98701-5_15.
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Bansal, Ankush, and Pulkit Anupam Srivastava. "Transcriptomics to Metabolomics." In Advances in Bioinformatics and Biomedical Engineering, 188–206. IGI Global, 2018. http://dx.doi.org/10.4018/978-1-5225-2607-0.ch008.
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A lot of omics data is generated in a recent decade which flooded the internet with transcriptomic, genomics, proteomics and metabolomics data. A number of software, tools, and web-servers have developed to analyze the big data omics. This review integrates the various methods that have been employed over the years to interpret the gene regulatory and metabolic networks. It illustrates random networks, scale-free networks, small world network, bipartite networks and other topological analysis which fits in biological networks. Transcriptome to metabolome network is of interest because of key enzymes identification and regulatory hub genes prediction. It also provides an insight into the understanding of omics technologies, generation of data and impact of in-silico analysis on the scientific community.
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Bansal, Ankush, and Pulkit Anupam Srivastava. "Transcriptomics to Metabolomics." In Biotechnology, 361–79. IGI Global, 2019. http://dx.doi.org/10.4018/978-1-5225-8903-7.ch014.
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A lot of omics data is generated in a recent decade which flooded the internet with transcriptomic, genomics, proteomics and metabolomics data. A number of software, tools, and web-servers have developed to analyze the big data omics. This review integrates the various methods that have been employed over the years to interpret the gene regulatory and metabolic networks. It illustrates random networks, scale-free networks, small world network, bipartite networks and other topological analysis which fits in biological networks. Transcriptome to metabolome network is of interest because of key enzymes identification and regulatory hub genes prediction. It also provides an insight into the understanding of omics technologies, generation of data and impact of in-silico analysis on the scientific community.
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Zhang, Wei, Gabriel R. Fries, and Joao Quevedo. "The Use of Bioinformatics and Big Data for the In Silico Study of Psychiatric Disorders." In Convergence Mental Health, edited by Laura M. Hack and Leanne M. Williams, 255–68. Oxford University Press, 2021. http://dx.doi.org/10.1093/med/9780197506271.003.0017.
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Mental and behavioral disorders are becoming the leading cause of disability across the world. Along with the ongoing development of biomedical and computational technologies, more and more data are being constantly produced, including genomic, transcriptomic, metabolomic, proteomic, clinical, and imaging resources. As a consequence, scientists in the psychiatric field are actively changing their research ways from studies focused on individual investigators to large international consortia, which accelerate the data accumulation and increase its size. This chapter discusses the current publicly available data sets on psychiatry disorders and neuroscience, as well as their integrated analysis. The authors also list some studies using novel types of data, which will further extent the potential of big data in the study of psychiatric disorders.
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Buy, Mélanie, William Digan, Xiaoyi Chen, Julien Husson, Mickael Ménager, Frédéric Rieux-Laucat, and Nicolas Garcelon. "A Multi-Omics Common Data Model for Primary Immunodeficiencies." In MEDINFO 2021: One World, One Health – Global Partnership for Digital Innovation. IOS Press, 2022. http://dx.doi.org/10.3233/shti220031.
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Primary Immunodeficiencies (PIDs) are associated with more than 400 rare monogenic diseases affecting various biological functions (e.g., development, regulation of the immune response) with a heterogeneous clinical expression (from no symptom to severe manifestations). To better understand PIDs, the ATRACTion project aims to perform a multi-omics analysis of PIDs cases versus a control group patients, including single-cell transcriptomics, epigenetics, proteomics, metabolomics, metagenomics and lipidomics. In this study, our goal is to develop a common data model integrating clinical and omics data, which can be used to obtain standardized information necessary for characterization of PIDs patients and for further systematic analysis. For that purpose, we extend the OMOP Common Data Model (CDM) and propose a multi-omics ATRACTion OMOP-CDM to integrate multi-omics data. This model, available for the community, is customizable for other types of rare diseases (https://framagit.org/imagine-plateforme-bdd/pub-rhu4-atraction).
Conference papers on the topic "Integrated proteomic analysi"
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Selvam, Anjan Panneer, and Shalini Prasad. "Single Molecule Analysis Tool (SMAT) for Multiplexed Label-Free Assessment of Rare Cell Populations." In ASME 2014 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2014. http://dx.doi.org/10.1115/imece2014-40225.
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A nanowell sensor for single molecular proteomic analysis of lung cancer has been designed. The nanowell sensor is an electrochemical immunoassay and comprises of a heterogenous nanoporous arrays integrated on to a gold microelectronic platform. The sensor operates on the principle of electrochemical impedance spectroscopy (EIS). Our approach to classification of lung cancer is based on screening for levels of expression of specific proteomic biomarkers associated with lung cancer stem cells. Proteomic activity for two lung cancer cell lines for two specific markers (ALDH1A1 and ALDH1A3) was quantified. Test samples prepared by synthetically spiking human pooled serum were tested and quantified for cancer stem cell marker activity. The lowest proteomic activity measured with (a) ALDH1A3 was 0.01 ng/mL and (b) ALDH1A1 was 1 ng/mL correlating to the detection of unit stem cell count.
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Aderogba, Samuel, J. Mark Meacham, F. Levent Degertekin, and Andrei G. Fedorov. "Micromachined Ultrasonic ElectroSpray Source Array for High Throughput Mass Spectrometry." In ASME 2004 3rd Integrated Nanosystems Conference. ASMEDC, 2004. http://dx.doi.org/10.1115/nano2004-46086.
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According to the recent Laboratory News’ Proteomics Special article Mass Spectroscopy (MS) has become the technology of choice to meet today’s unprecedented demand for accurate bioanalytical measurements, including protein identification. Although MS can be used to analyze any biological sample, it must be first converted to gas-phase ions before it can be introduced into a mass spectrometer for analysis. It is transfer of a very small liquid sample (proteins are very expensive and often very difficult to produce in sizable quantities) into a gas-phase ions that is currently considered to be a bottleneck to high throughput proteomics. Electrospray ionization (ESI) is a technique developed in early 1990th to generate a spray gas-phase ions by applying high voltage (from several hundreds volts and up to a few thousands kilovolts relative to the ground electrode of the MS interface) to a small capillary through which the liquid solution is pumped. The high electric field ionizes the fluid forming the converging Taylor cone of the exiting jet which eventually breaks into many small droplets when the repulsive Coulombic forces overcome the surface tension. Because of the focusing effect associated with the spraying the electrically charged fluid, the size of the electrospray cone and thus of the formed droplets is in a few tens of nanometers range although the inner diameter of the capillary is in the micrometer range.
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Kalaitzakis, Manos, Vangelis Kritsotakis, Haridimos Kondylakis, George Potamias, Manolis Tsiknakis, and Dimitris Kafetzopoulos. "An Integrated Clinico-Proteomics Information Management and Analysis Platform." In 2008 21st International Symposium on Computer-Based Medical Systems (CBMS). IEEE, 2008. http://dx.doi.org/10.1109/cbms.2008.48.
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Zhong, Alex, Alice Liu, and Amy Wu. "CoFIM: A Computational Framework for Proteomic and Metabolomic Integrated Data Analysis." In DMIP '21: 2021 4th International Conference on Digital Medicine and Image Processing. New York, NY, USA: ACM, 2021. http://dx.doi.org/10.1145/3506651.3506658.
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Liu, Jikun, Chien-Fu Chen, Chien-Cheng Chang, and Don L. DeVoe. "Isoelectric Focusing-Reversed Phase Liquid Chromatography Polymer Microchip With Integrated High-Pressure Valves." In ASME 2009 International Mechanical Engineering Congress and Exposition. ASMEDC, 2009. http://dx.doi.org/10.1115/imece2009-12147.
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A cyclicolefin polymer (COP) microchip supporting parallel 2-D peptide separations is described. By combining isoelectric focusing (IEF) as a first dimension and parallel reversed-phase liquid chromatography (RPLC) as a second dimension, the system enables efficient high-throughput fractionation prior to mass spectrometry in support of peptide mass fingerprinting for global proteomic analysis from highly limited specimens. The IEF-RPLC chip incorporates high-pressure micro shut-off valves, allowing uniform sample transfer and gradient elution from each micro LC column, and ensuring hydrodynamic isolation between the separation dimensions. The utility of the initial microchip is demonstrated by separation of a fluorescein labeled bovine serum albumin tryptic digest in a chip containing a five channel RPLC array.
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Dingerdissen, Hayley, and Raja Mazumder. "Abstract 4875: HIVE Proteomics: Integrated, cloud-based RNA-Seq and proteomics analysis of prostate adenocarcinoma samples." In Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA. American Association for Cancer Research, 2015. http://dx.doi.org/10.1158/1538-7445.am2015-4875.
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Moran, Michael F., Christine To, Yuhong Wei, Lei Li, Paul Taylor, Vladimir Ignatchenko, Dan Strumpf, et al. "Abstract 5127: Characterization of lung cancers by integrated genomic and proteomic analysis." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-5127.
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Tsiknakis, M., P. Grangeat, P.-A. Binz, G. Potamias, F. Lisacek, L. Gerfault, C. Paulus, et al. "Functional specifications of an integrated proteomics information management and analysis platform." In 2007 29th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE, 2007. http://dx.doi.org/10.1109/iembs.2007.4353732.
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Lee, HeaYeon, and JuKyung Lee. "Advanced Biomimetic Nanodevice Using Nanotechnology Addressable Lipid Rafts Nanoarrays Toward Advanced Nanomaterials." In ASME 2013 2nd Global Congress on NanoEngineering for Medicine and Biology. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/nemb2013-93286.
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In recent years, a new paradigm of nanobiomedical devices combining miniaturization and integration has been exploited in areas such as combinational chemistry, biotechnology, engineering, proteomics and clinical diagnostics. One of the critical issues in the development of nanobiomedical system is how to differentiate signal-to-noise ratio per very small amount of signal. Biocompatible integrated nanopattern requires the fabrication of appropriately designed nanomatrix for high sensitivity homogenous assays, which are capable of ultimately mimic the physiological environment. We reported the nanomatrix geometry of a well-oriented nanowell array derived from nanofabrication technology which can easily be employed for digital detection with a high S/N ratio, miniaturization, integrated assays and single molecule analysis. In this present, we describe a nano(submicro) array of tethered lipid bilayer raft membranes comprising a biosensing platform.
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Chu, Ching-Yu, Szu-Yuan Chen, Fu-Yu Chueh, Mei-Ling Cheng, and Chao-Lan Yu. "Abstract 2564: Integrated transcriptomic, proteomic, and metabolomic analyses of human and mouse T cell leukemia." In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-2564.
Full textReports on the topic "Integrated proteomic analysi"
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Heifetz, Yael, and Michael Bender. Success and failure in insect fertilization and reproduction - the role of the female accessory glands. United States Department of Agriculture, December 2006. http://dx.doi.org/10.32747/2006.7695586.bard.
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The research problem. Understanding of insect reproduction has been critical to the design of insect pest control strategies including disruptions of mate-finding, courtship and sperm transfer by male insects. It is well known that males transfer proteins to females during mating that profoundly affect female reproductive physiology, but little is known about the molecular basis of female mating response and no attempts have yet been made to interfere with female post-mating responses that directly bear on the efficacy of fertilization. The female reproductive tract provides a crucial environment for the events of fertilization yet thus far those events and the role of the female tract in influencing them are poorly understood. For this project, we have chosen to focus on the lower reproductive tract because it is the site of two processes critical to reproduction: sperm management (storage, maintenance, and release from storage) and fertilization. E,fforts during this project period centered on the elucidation of mating responses in the female lower reproductive tract The central goals of this project were: 1. To identify mating-responsive genes in the female lower reproductive tract using DNA microarray technology. 2. In parallel, to identify mating-responsive genes in these tissues using proteomic assays (2D gels and LC-MS/MS techniques). 3. To integrate proteomic and genomic analyses of reproductive tract gene expression to identify significant genes for functional analysis. Our main achievements were: 1. Identification of mating-responsive genes in the female lower reproductive tract. We identified 539 mating-responsive genes using genomic and proteomic approaches. This analysis revealed a shift from gene silencing to gene activation soon after mating and a peak in differential gene expression at 6 hours post-mating. In addition, comparison of the two datasets revealed an expression pattern consistent with the model that important reproductive proteins are pre-programmed for synthesis prior to mating. This work was published in Mack et al. (2006). Validation experiments using real-time PCR techniques suggest that microarray assays provide a conservativestimate of the true transcriptional activity in reproductive tissues. 2.lntegration of proteomics and genomics data sets. We compared the expression profiles from DNA microarray data with the proteins identified in our proteomic experiments. Although comparing the two data sets poses analyical challenges, it provides a more complete view of gene expression as well as insights into how specific genes may be regulated. This work was published in Mack et al. (2006). 3. Development of primary reproductive tract cell cultures. We developed primary cell cultures of dispersed reproductive tract cell types and determined conditions for organ culture of the entire reproductive tract. This work will allow us to rapidly screen mating-responsive genes for a variety of reproductive-tract specifi c functions. Scientific and agricultural significance. Together, these studies have defined the genetic response to mating in a part of the female reproductive tract that is critical for successful fertllization and have identified alarge set of mating-responsive genes. This work is the first to combine both genomic and proteomic approaches in determining female mating response in these tissues and has provided important insights into insect reproductive behavior.
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Schaffer, Arthur A., and Jocelyn Rose. Understanding Cuticle Development in Tomato through the Study of Novel Germplasm with Malformed Cuticles. United States Department of Agriculture, June 2013. http://dx.doi.org/10.32747/2013.7593401.bard.
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Plant cuticle development and metabolism are still poorly understood, partly due to the chemical complexity of the cuticular layer. The overall research objective was to broaden and deepen our understanding of tomato fruit cuticle development by analyzing novel germplasm with cuticular malformations and by studying the transcriptome and proteome of the fruit epidermal tissues, as strategies to overcome the challenges posed by the recalcitrance of the biological system. During the project we succeeded in identifying two genes with major impact on cuticle development. One of these encoded the first cutin synthase to be identified in plants, a metabolic step that had been a black box in cutin synthesis. In addition genes controlling the triterpenoid components of the cuticle were identified and, most interestingly, genetic variability for this component was identified among the wild tomato species germplasm. Additional germplasm was developed based on interspecific crosses that will allow for the future characterization of modifier genes that interact with the microfissuring gene (CWP) to promote or inhibit fruit cracking. One of the major accomplishments of the joint project was the integrated transcriptomic and proteomic analysis of the fruit cuticle and underlying tissues which allows for the identification of the pericarp cell layers responsible for the extracellular, cuticle-localized protein component. The results of the project have expanded our understanding of tomato fruit cuticle development and its genetic control. In addition, germplasm developed will be useful in developing tomato varieties resistant to cracking, on the one hand, and varieties useful for the dehydration industry on the other.
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Blumwald, Eduardo, and Avi Sadka. Citric acid metabolism and mobilization in citrus fruit. United States Department of Agriculture, October 2007. http://dx.doi.org/10.32747/2007.7587732.bard.
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Accumulation of citric acid is a major determinant of maturity and fruit quality in citrus. Many citrus varieties accumulate citric acid in concentrations that exceed market desires, reducing grower income and consumer satisfaction. Citrate is accumulated in the vacuole of the juice sac cell, a process that requires both metabolic changes and transport across cellular membranes, in particular, the mitochondrial and the vacuolar (tonoplast) membranes. Although the accumulation of citrate in the vacuoles of juice cells has been clearly demonstrated, the mechanisms for vacuolar citrate homeostasis and the components controlling citrate metabolism and transport are still unknown. Previous results in the PIs’ laboratories have indicated that the expression of a large number of a large number of proteins is enhanced during fruit development, and that the regulation of sugar and acid content in fruits is correlated with the differential expression of a large number of proteins that could play significant roles in fruit acid accumulation and/or regulation of acid content. The objectives of this proposal are: i) the characterization of transporters that mediate the transport of citrate and determine their role in uptake/retrieval in juice sac cells; ii) the study of citric acid metabolism, in particular the effect of arsenical compounds affecting citric acid levels and mobilization; and iii) the development of a citrus fruit proteomics platform to identify and characterize key processes associated with fruit development in general and sugar and acid accumulation in particular. The understanding of the cellular processes that determine the citrate content in citrus fruits will contribute to the development of tools aimed at the enhancement of citrus fruit quality. Our efforts resulted in the identification, cloning and characterization of CsCit1 (Citrus sinensis citrate transporter 1) from Navel oranges (Citrus sinesins cv Washington). Higher levels of CsCit1 transcripts were detected at later stages of fruit development that coincided with the decrease in the juice cell citrate concentrations (Shimada et al., 2006). Our functional analysis revealed that CsCit1 mediates the vacuolar efflux of citrate and that the CsCit1 operates as an electroneutral 1CitrateH2-/2H+ symporter. Our results supported the notion that it is the low permeable citrateH2 - the anion that establishes the buffer capacity of the fruit and determines its overall acidity. On the other hand, it is the more permeable form, CitrateH2-, which is being exported into the cytosol during maturation and controls the citrate catabolism in the juice cells. Our Mass-Spectrometry-based proteomics efforts (using MALDI-TOF-TOF and LC2- MS-MS) identified a large number of fruit juice sac cell proteins and established comparisons of protein synthesis patterns during fruit development. So far, we have identified over 1,500 fruit specific proteins that play roles in sugar metabolism, citric acid cycle, signaling, transport, processing, etc., and organized these proteins into 84 known biosynthetic pathways (Katz et al. 2007). This data is now being integrated in a public database and will serve as a valuable tool for the scientific community in general and fruit scientists in particular. Using molecular, biochemical and physiological approaches we have identified factors affecting the activity of aconitase, which catalyze the first step of citrate catabolism (Shlizerman et al., 2007). Iron limitation specifically reduced the activity of the cytosolic, but not the mitochondrial, aconitase, increasing the acid level in the fruit. Citramalate (a natural compound in the juice) also inhibits the activity of aconitase, and it plays a major role in acid accumulation during the first half of fruit development. On the other hand, arsenite induced increased levels of aconitase, decreasing fruit acidity. We have initiated studies aimed at the identification of the citramalate biosynthetic pathway and the role(s) of isopropylmalate synthase in this pathway. These studies, especially those involved aconitase inhibition by citramalate, are aimed at the development of tools to control fruit acidity, particularly in those cases where acid level declines below the desired threshold. Our work has significant implications both scientifically and practically and is directly aimed at the improvement of fruit quality through the improvement of existing pre- and post-harvest fruit treatments.
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Manulis-Sasson, Shulamit, Christine D. Smart, Isaac Barash, Laura Chalupowicz, Guido Sessa, and Thomas J. Burr. Clavibacter michiganensis subsp. michiganensis-tomato interactions: expression and function of virulence factors, plant defense responses and pathogen movement. United States Department of Agriculture, February 2015. http://dx.doi.org/10.32747/2015.7594405.bard.
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Clavibactermichiganensissubsp. michiganensis(Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The goal of the project was to unravel the molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato. The genome of Cmm contains numerous genes encoding for extracellular serine proteases and cell wall degrading enzymes. The first objective was to elucidate the role of secreted serine proteases in Cmm virulence. Mutants of nine genes encoding serine proteases of 3 different families were tested for their ability to induce wilting, when tomato stems were puncture-inoculated, as compared to blisters formation on leaves, when plants were spray-inoculated. All the mutants showed reduction in wilting and blister formation as compared to the wild type. The chpCmutant displayed the highest reduction, implicating its major role in symptom development. Five mutants of cell wall degrading enzymes and additional genes (i.e. perforin and sortase) caused wilting but were impaired in their ability to form blisters on leaves. These results suggest that Cmm differentially expressed virulence genes according to the site of penetration. Furthermore, we isolated and characterized two Cmmtranscriptional activators, Vatr1 and Vatr2 that regulate the expression of virulence factors, membrane and secreted proteins. The second objective was to determine the effect of bacterial virulence genes on movement of Cmm in tomato plants and identify the routes by which the pathogen contaminates seeds. Using a GFP-labeledCmm we could demonstrate that Cmm extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem and formed biofilm-like structures composed of large bacterial aggregates. Our findings suggest that virulence factors located on the chp/tomAPAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates. We constructed a transposon plasmid that can be stably integrated into Cmm chromosome and express GFP, in order to follow movement to the seeds. Field strains from New York that were stably transformed with this construct, could not only access seeds systemically through the xylem, but also externally through tomato fruit lesions, which harbored high intra-and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruit began to ripen. These results highlight the ability of Cmm to invade tomato fruit and seed through multiple entry routes. The third objective was to assess correlation between disease severity and expression levels of Cmm virulence genes and tomato defense genes. The effect of plant age on expression of tomato defense related proteins during Cmm infection was analyzed by qRT-PCR. Five genes out of eleven showed high induction at early stages of infection of plants with 19/20 leaves compared to young plants bearing 7/8 leaves. Previous results showed that Cmm virulence genes were expressed at early stages of infection in young plants compared to older plants. Results of this study suggest that Cmm virulence genes may suppress expression of tomato defense-related genes in young plants allowing effective disease development. The possibility that chpCis involved in suppression of tomato defense genes is currently under investigation by measuring the transcript level of several PR proteins, detected previously in our proteomics study. The fourth objective was to define genome location and stability of virulence genes in Cmm strains. New York isolates were compared to Israeli, Serbian, and NCPPB382 strains. The plasmid profiles of New York isolates were diverse and differed from both Israeli and Serbian strains. PCR analysis indicated that the presence of putative pathogenicity genes varied between isolates and highlighted the ephemeral nature of pathogenicity genes in field populations of Cmm. Results of this project significantly contributed to the understanding of Cmm virulence, its movement within tomato xylem or externally into the seeds, the role of serine proteases in disease development and initiated research on global regulation of Cmm virulence. These results form a basis for developing new strategies to combat wilt and canker disease of tomato.