WorldWideScience

Sample records for cell proteome perspectives

  1. STEM CELLS AND PROTEOMICS

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yong-ming; GUO Tian-nan; HUANG Shi-ang

    2006-01-01

    The distinctive features of proteomics are large-scale and high throughput. The key techniques of proteomics are two-dimensional gel electrophoresis, mass spectrometry and bioinformatics. Stem cell can differentiate into all kinds of cells, tissues and organs. There are many proteins and cytokines involved in the process of differentiation. Applying proteomics techniques to the research of the complex process of stem cell differentiation is of great importance to study the mechanism and applications of stem cell differentiation.

  2. Effects of 5'-azacytidine and alendronate on a hepatocellular carcinoma cell line: a proteomics perspective.

    Science.gov (United States)

    Ilyas, Amber; Hashim, Zehra; Zarina, Shamshad

    2015-07-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer related deaths around the world. Due to late diagnosis and development of drug resistance in patients suffering from HCC, development of more effective therapeutic strategies is inevitable. The aim of this study was to evaluate the combined apoptotic effect of 5'-Azacytidine (5'-AzaC) and alendronate (ALN) on Huh-7 HCC cell line and to explore differential expression at genomics and proteomics level. Incubation of HCC cell line with 5'-AzaC alone showed cell death in a time and dose dependent manner while in combination with ALN, increased cytotoxicity was observed. Up-regulation of CASP7(Caspase7) and LZTS1 (leucine zipper, putative tumor suppressor 1) and down-regulation of DNMT1(DNA (cytosine-5-)-methyltransferase 1) was noted in treated cells. Proteomic studies on the treated cells revealed altered expression of different proteins including peroxiredoxin 2 (Prx2), Annexin 5 (Anx5), Rho GTPase activating protein (RhoGAP), Nuclear factor-kappa B (NF-kB), tumor necrosis factor alpha-induced protein (TNF), triosephosphate isomerase (TPI), Glutathione S transferase (GSTP1) and Heat shock protein60 (HSP60). Our study demonstrated the cytotoxic effect of 5'-AzaC and ALN drug combination on Huh-7 HCC cells suggesting such combinations may be explored as a possible therapeutic approach. Current study revealed that Huh-7 HCC cells are sensitive to 5'-AzaC and ALN drug combination and such combination approaches could lead to the development of new therapeutic strategies. Furthermore, we also report the expression of Anx5 exclusively in untreated cancerous cell line indicating the possibility of being used as a potential therapeutic target and biomarker. PMID:25854900

  3. A proteomic perspective on the changes in milk proteins due to high somatic cell count.

    Science.gov (United States)

    Zhang, L; Boeren, S; van Hooijdonk, A C M; Vervoort, J M; Hettinga, K A

    2015-08-01

    Although cows with subclinical mastitis have no difference in the appearance of their milk, milk composition and milk quality are altered because of the inflammation. To know the changes in milk quality with different somatic cell count (SCC) levels, 5 pooled bovine milk samples with SCC from 10(5) to 10(6) cells/mL were analyzed qualitatively and quantitatively using both one-dimension sodium dodecyl sulfate PAGE and filter-aided sample preparation coupled with dimethyl labeling, both followed by liquid chromatography tandem mass spectrometry. Minor differences were found on the qualitative level in the proteome from milk with different SCC levels, whereas the concentration of milk proteins showed remarkable changes. Not only immune-related proteins (cathelicidins, IGK protein, CD59 molecule, complement regulatory protein, lactadherin), but also proteins with other biological functions (e.g., lipid metabolism: platelet glycoprotein 4, butyrophilin subfamily 1 member A1, perilipin-2) were significantly different in milk from cows with high SCC level compared with low SCC level. The increased concentration of protease inhibitors in the milk with higher SCC levels may suggest a protective role in the mammary gland against protease activity. Prostaglandin-H2 D-isomerase showed a linear relation with SCC, which was confirmed with an ELISA. However, the correlation coefficient was lower in individual cows compared with bulk milk. These results indicate that prostaglandin-H2 D-isomerase may be used as an indicator to evaluate bulk milk quality and thereby reduce the economic loss in the dairy industry. The results from this study reflect the biological phenomena occurring during subclinical mastitis and in addition provide a potential indicator for the detection of bulk milk with high SCC. PMID:26094216

  4. Translational plant proteomics: A perspective

    NARCIS (Netherlands)

    Agrawal, G.K.; Pedreschi, R.; Barkla, B.J.; Bindschedler, L.V.; Cramer, R.; Sarkar, A.; Renaut, J.; Job, D.; Rakwal, R.

    2012-01-01

    Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic v

  5. Proteomics of cell-cell interactions in health and disease.

    Science.gov (United States)

    Lindoso, Rafael S; Sandim, Vanessa; Collino, Federica; Carvalho, Adriana B; Dias, Juliana; da Costa, Milene R; Zingali, Russolina B; Vieyra, Adalberto

    2016-01-01

    The mechanisms of cell-cell communications are now under intense study by proteomic approaches. Proteomics has unraveled changes in protein profiling as the result of cell interactions mediated by ligand/receptor, hormones, soluble factors, and the content of extracellular vesicles. Besides being a brief overview of the main and profitable methodologies now available (evaluating theory behind the methods, their usefulness, and pitfalls), this review focuses on-from a proteome perspective-some signaling pathways and post-translational modifications (PTMs), which are essential for understanding ischemic lesions and their recovery in two vital organs in mammals, the heart, and the kidney. Knowledge of misdirection of the proteome during tissue recovery, such as represented by the convergence between fibrosis and cancer, emerges as an important tool in prognosis. Proteomics of cell-cell interaction is also especially useful for understanding how stem cells interact in injured tissues, anticipating clues for rational therapeutic interventions. In the effervescent field of induced pluripotency and cell reprogramming, proteomic studies have shown what proteins from specialized cells contribute to the recovery of infarcted tissues. Overall, we conclude that proteomics is at the forefront in helping us to understand the mechanisms that underpin prevalent pathological processes. PMID:26552723

  6. Cell death proteomics database: consolidating proteomics data on cell death.

    Science.gov (United States)

    Arntzen, Magnus Ø; Bull, Vibeke H; Thiede, Bernd

    2013-05-01

    Programmed cell death is a ubiquitous process of utmost importance for the development and maintenance of multicellular organisms. More than 10 different types of programmed cell death forms have been discovered. Several proteomics analyses have been performed to gain insight in proteins involved in the different forms of programmed cell death. To consolidate these studies, we have developed the cell death proteomics (CDP) database, which comprehends data from apoptosis, autophagy, cytotoxic granule-mediated cell death, excitotoxicity, mitotic catastrophe, paraptosis, pyroptosis, and Wallerian degeneration. The CDP database is available as a web-based database to compare protein identifications and quantitative information across different experimental setups. The proteomics data of 73 publications were integrated and unified with protein annotations from UniProt-KB and gene ontology (GO). Currently, more than 6,500 records of more than 3,700 proteins are included in the CDP. Comparing apoptosis and autophagy using overrepresentation analysis of GO terms, the majority of enriched processes were found in both, but also some clear differences were perceived. Furthermore, the analysis revealed differences and similarities of the proteome between autophagosomal and overall autophagy. The CDP database represents a useful tool to consolidate data from proteome analyses of programmed cell death and is available at http://celldeathproteomics.uio.no. PMID:23537399

  7. Cell wall proteomics of crops

    OpenAIRE

    Komatsu, Setsuko; Yanagawa, Yuki

    2013-01-01

    Cell wall proteins play key roles in cell structure and metabolism, cell enlargement, signal transduction, responses to environmental stress, and many other physiological events. Agricultural crops are often used for investigating stress tolerance because cultivars with differing degrees of tolerance are available. Abiotic and biotic stress factors markedly influence the geographical distribution and yields of many crop species. Crop cell wall proteomics is of particular importance for improv...

  8. A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation

    Directory of Open Access Journals (Sweden)

    Tedeschi Lorena

    2010-03-01

    Full Text Available Abstract Background The use of chromatography coupled with mass spectrometry (MS analysis is a powerful approach to identify proteins, owing to its capacity to fractionate molecules according to different chemical features. The first protein expression map of vascular smooth muscle cells (VSMC was published in 2001 and since then other papers have been produced. The most detailed two-dimensional polyacrylamide gel electrophoresis (2D-PAGE map was presented by Mayr et al who identified 235 proteins, corresponding to the 154 most abundant unique proteins in mouse aortic VSMC. A chromatographic approach aimed at fractionating the VSMC proteome has never been used before. Results This paper describes a strategy for the study of the VSMC proteome. Our approach was based on pre-fractionation with ion exchange chromatography coupled with matrix assisted laser desorption-time of flight mass spectrometry analysis assisted by a liquid chromatography (LC-MALDI-TOF/TOF. Ion exchange chromatography resulted in a good strategy designed to simplify the complexity of the cellular extract and to identify a large number of proteins. Selectivity based on the ion-exchange chemical features was adequate if evaluated on the basis of protein pI. The LC-MALDI approach proved to be highly reproducible and sensitive since we were able to identify up to 815 proteins with a concentration dynamic range of 7 orders of magnitude. Conclusions In our opinion, the large number of identified proteins and the promising quantitative reproducibility made this approach a powerful method to analyze complex protein mixtures in a high throughput way and to obtain statistical data for the discovery of key factors involved in VSMC activation and to analyze a label-free differential protein expression.

  9. Proteomics Study of Cotton Fiber Cells

    Institute of Scientific and Technical Information of China (English)

    LIU Jin-yuan

    2008-01-01

    @@ A comparative proteomic analysis was applied to explore the mechanism of fiber cell development in cotton.Initially,an efficient protein preparation method was established for proteomic analysis of developing cotton fibers by two-dimensional gel electrophoresis,and a microwave enhanced ink staining technique also was created for fast and sensitive protein quantification in proteomic studies.

  10. Proteomic profiling of exosomes: Current perspectives

    DEFF Research Database (Denmark)

    Simpson, Richard J; Jensen, Søren S; Lim, Justin W E

    2008-01-01

    Exosomes are 40-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro. Recent studies have shown that exosomes are also found in vivo in body fluids such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk....... While the biological function of exosomes is still unclear, they can mediate communication between cells, facilitating processes such as antigen presentation and in trans signaling to neighboring cells. Exosome-like vesicles identified in Drosophila (referred to as argosomes) may be potential vehicles...... for the spread of morphogens in epithelia. The advent of current MS-based proteomic technologies has contributed significantly to our understanding of the molecular composition of exosomes. In addition to a common set of membrane and cytosolic proteins, it is becoming increasingly apparent that exosomes harbor...

  11. New challenges for proteomics technologies: a mini perspective review

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yufeng; Pasa-Tolic, Ljiljana; Robinson, Errol W.; Adkins, Joshua N.; Smith, Richard D.

    2014-10-10

    Proteomics technologies have experienced rapid advances over the last decade to identify or quantify thousands of proteins per sample, typically in a few hours, enabling proteomics applications in environmental, biological, medical, and clinical research. A number of publications have reviewed advances in proteomic technologies and applications. This short review focuses first on a discussion of sensitivity in bottom-up (i.e. digested protein) proteomics and approaches for characterization of small cell populations, and secondly on protein separations for top-down (i.e. intact protein) proteomics including discussions of key technical challenges where recent advances are elucidating specific functions of proteins in biological processes.

  12. Proteomic Analysis of Chinese Hamster Ovary Cells

    DEFF Research Database (Denmark)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan; Nagarajan, Harish; Sarkaria, Vishaldeep; Kumar, Amit; Wolozny, Daniel; Colao, Joe; Jacobson, Elena; Tian, Yuan; O’Meally, Robert N.; Krag, Sharon S.; Cole, Robert N.; Palsson, Bernhard; Zhang, Hui; Betenbaugh, Michael

    2012-01-01

    this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...... identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From...

  13. Proteomics

    DEFF Research Database (Denmark)

    Tølbøll, Trine Højgaard; Danscher, Anne Mette; Andersen, Pia Haubro;

    2012-01-01

    grouped manually to one or more of five major functional groups related to metabolism, cell structure, immunity, apoptosis and angiogenesis. These were chosen to represent basic cell functions and biological processes potentially involved in the pathogenesis of CHD. The LC–MS/MS-based proteomic analysis...... presented here is the largest published survey, so far, of the bovine claw tissue proteome....

  14. Proteomic analysis of Chinese hamster ovary cells.

    Science.gov (United States)

    Baycin-Hizal, Deniz; Tabb, David L; Chaerkady, Raghothama; Chen, Lily; Lewis, Nathan E; Nagarajan, Harish; Sarkaria, Vishaldeep; Kumar, Amit; Wolozny, Daniel; Colao, Joe; Jacobson, Elena; Tian, Yuan; O'Meally, Robert N; Krag, Sharon S; Cole, Robert N; Palsson, Bernhard O; Zhang, Hui; Betenbaugh, Michael

    2012-11-01

    To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions. PMID:22971049

  15. Neural Stem Cells (NSCs) and Proteomics.

    Science.gov (United States)

    Shoemaker, Lorelei D; Kornblum, Harley I

    2016-02-01

    Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. PMID:26494823

  16. Neural Stem Cells (NSCs) and Proteomics*

    Science.gov (United States)

    Shoemaker, Lorelei D.; Kornblum, Harley I.

    2016-01-01

    Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. PMID:26494823

  17. Top Down proteomics: Facts and perspectives

    International Nuclear Information System (INIS)

    Highlights: • Top Down versus Bottom Up proteomics analysis. • Separations methods for Top Down proteomics. • Developments in mass spectrometry instrumentation and fragmentation. • Native mass spectrometry. - Abstract: The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years

  18. Top Down proteomics: Facts and perspectives

    Energy Technology Data Exchange (ETDEWEB)

    Catherman, Adam D.; Skinner, Owen S.; Kelleher, Neil L., E-mail: n-kelleher@northwestern.edu

    2014-03-21

    Highlights: • Top Down versus Bottom Up proteomics analysis. • Separations methods for Top Down proteomics. • Developments in mass spectrometry instrumentation and fragmentation. • Native mass spectrometry. - Abstract: The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

  19. Proteomics

    DEFF Research Database (Denmark)

    Dam, Svend; Stougaard, Jens

    2014-01-01

    Proteomics is an efficient tool to identify proteins present in specific tissues, cell types, or organelles. The resulting proteome reference maps and/or comparative analyses provide overviews of regulated proteins between wild type and mutants or between different conditions together with a...... comprehensive list of proteins. Post translation modifications (PTMs), such as glycosylation and phosphorylation, are pivotal for protein stability and function. Several strategies for enrichment of PTMs have been developed where targeted proteomic approaches are used to identify these PTMs. The sequenced and...... annotated Lotus japonicus (Lotus) genome has been essential for obtaining high-quality protein identifications from proteomics studies. Furthermore, additional genomics and transcriptomics studies from several Lotus species/ecotypes support putative gene structures and these can be further supported using...

  20. Proteomics perspectives in rotator cuff research

    DEFF Research Database (Denmark)

    Sejersen, Maria Hee Jung; Frost, Poul; Hansen, Torben Bæk;

    2015-01-01

    Background Rotator cuff tendinopathy including tears is a cause of significant morbidity. The molecular pathogenesis of the disorder is largely unknown. This review aimed to present an overview of the literature on gene expression and protein composition in human rotator cuff tendinopathy and other...... studies on objectively quantified differential gene expression and/or protein composition in human rotator cuff tendinopathy and other tendinopathies as compared to control tissue. Results We identified 2199 studies, of which 54 were included; 25 studies focussed on rotator cuff or biceps tendinopathy......, which only allowed simultaneous quantification of a limited number of prespecified mRNA molecules or proteins, several proteins appeared to be differentially expressed/represented in rotator cuff tendinopathy and other tendinopathies. No proteomics studies fulfilled our inclusion criteria, although...

  1. The pancreatic beta cell surface proteome

    OpenAIRE

    Stützer, I.; Esterházy, D.; Stoffel, M.

    2012-01-01

    The pancreatic beta cell is responsible for maintaining normoglycaemia by secreting an appropriate amount of insulin according to blood glucose levels. The accurate sensing of the beta cell extracellular environment is therefore crucial to this endocrine function and is transmitted via its cell surface proteome. Various surface proteins that mediate or affect beta cell endocrine function have been identified, including growth factor and cytokine receptors, transporters, ion channels and prote...

  2. Membrane proteomic analysis of pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Liu Xiaojun

    2010-09-01

    Full Text Available Abstract Background Pancreatic cancer is one of the most aggressive human tumors due to its high potential of local invasion and metastasis. The aim of this study was to characterize the membrane proteomes of pancreatic ductal adenocarcinoma (PDAC cells of primary and metastatic origins, and to identify potential target proteins related to metastasis of pancreatic cancer. Methods Membrane/membrane-associated proteins were isolated from AsPC-1 and BxPC-3 cells and identified with a proteomic approach based on SDS-PAGE, in-gel tryptic digestion and liquid chromatography with tandem mass spectrometry (LC-MS/MS. X! Tandem was used for database searching against the SwissProt human protein database. Results We identified 221 & 208 proteins from AsPC-1 and BxPC-3 cells, respectively, most of which are membrane or membrane-associated proteins. A hundred and nine proteins were found in both cell lines while the others were present in either AsPC-1 or BxPC-3 cells. Differentially expressed proteins between two cell lines include modulators of cell adhesion, cell motility or tumor invasion as well as metabolic enzymes involved in glycolysis, tricarboxylic acid cycle, or nucleotide/lipid metabolism. Conclusion Membrane proteomes of AsPC-1 (metastatic and BxPC-3 (primary cells are remarkably different. The differentially expressed membrane proteins may serve as potential targets for diagnostic and therapeutic interventions.

  3. Proteomic cornerstones of hematopoietic stem cell differentiation

    DEFF Research Database (Denmark)

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon;

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors...... which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem....../progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5,000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical...

  4. Quantitative Proteomics Analysis of Leukemia Cells.

    Science.gov (United States)

    Halbach, Sebastian; Dengjel, Jörn; Brummer, Tilman

    2016-01-01

    Chronic myeloid leukemia (CML) is driven by the oncogenic fusion kinase Bcr-Abl, which organizes its own signaling network with various proteins. These proteins, their interactions, and their role in relevant signaling pathways can be analyzed by quantitative mass spectrometry (MS) approaches in various models systems, e.g., in cell culture models. In this chapter, we describe in detail immunoprecipitations and quantitative proteomics analysis using stable isotope labeling by amino acids in cell culture (SILAC) of components of the Bcr-Abl signaling pathway in the human CML cell line K562. PMID:27581145

  5. Proteomics of neural stem cells

    Czech Academy of Sciences Publication Activity Database

    Skalníková, Helena; Vodička, Petr; Gadher, S. J.; Kovářová, Hana

    2008-01-01

    Roč. 5, č. 2 (2008), s. 175-186. ISSN 1478-9450 R&D Projects: GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z50450515 Keywords : cell-based regnerative and reparative therapy * conditioned media * differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.848, year: 2008

  6. Cell wall proteins: a new insight through proteomics

    OpenAIRE

    Jamet, Elisabeth; Canut, Hervé; Boudart, Georges; Pont-Lezica, Rafael F

    2006-01-01

    Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translation...

  7. Mass spectrometry-based proteomics in cell biology

    OpenAIRE

    Walther, T. C.; Mann, M

    2010-01-01

    The global analysis of protein composition, modifications, and dynamics are important goals in cell biology. Mass spectrometry (MS)–based proteomics has matured into an attractive technology for this purpose. Particularly, high resolution MS methods have been extremely successful for quantitative analysis of cellular and organellar proteomes. Rapid advances in all areas of the proteomic workflow, including sample preparation, MS, and computational analysis, should make the technology more eas...

  8. Proteomes and Neural Stem Cells: cellular signalling during differentiation

    Czech Academy of Sciences Publication Activity Database

    Skalníková, Helena; Halada, Petr; Vodička, Petr; Motlík, Jan; Horning, O.; Jensen, O. N.; Gadher, S. J.; Pelech, S.; Kovářová, Hana

    Cambridge : -, 2007, s. 1-1. [BSPR-EBI Meeting: Integrative Proteomics: From Molecules to Systems,. Cambridge (GB), 25.07.2007-27.07.2007] Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : neural stem cells * differentiation * signalling * proteome Subject RIV: EB - Genetics ; Molecular Biology

  9. Clinical proteomics-driven precision medicine for targeted cancer therapy: current overview and future perspectives.

    Science.gov (United States)

    Zhou, Li; Wang, Kui; Li, Qifu; Nice, Edouard C; Zhang, Haiyuan; Huang, Canhua

    2016-04-01

    Cancer is a common disease that is a leading cause of death worldwide. Currently, early detection and novel therapeutic strategies are urgently needed for more effective management of cancer. Importantly, protein profiling using clinical proteomic strategies, with spectacular sensitivity and precision, offer excellent promise for the identification of potential biomarkers that would direct the development of targeted therapeutic anticancer drugs for precision medicine. In particular, clinical sample sources, including tumor tissues and body fluids (blood, feces, urine and saliva), have been widely investigated using modern high-throughput mass spectrometry-based proteomic approaches combined with bioinformatic analysis, to pursue the possibilities of precision medicine for targeted cancer therapy. Discussed in this review are the current advantages and limitations of clinical proteomics, the available strategies of clinical proteomics for the management of precision medicine, as well as the challenges and future perspectives of clinical proteomics-driven precision medicine for targeted cancer therapy. PMID:26923776

  10. Proteomics Applied to Porcine and Human Neural Stem Cell Differentiation

    Czech Academy of Sciences Publication Activity Database

    Mairychová, Kateřina; Skalníková, Helena; Tylečková, Jiřina; Halada, Petr; Marsala, M.; Kovářová, Hana

    Liběchov : Institute of Animal Physiology and Genetics AS CR, v.v.i, 2010. s. 61-61. [Informal Proteomic Meeting 2010. 09.11.2010-10.11.2010, Liblice] R&D Projects: GA MŠk 1M0538; GA MŠk(CZ) ME10044 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : proteomics * cell differentiation * neural stem cell s Subject RIV: FH - Neurology

  11. Human Pituitary Adenoma Proteomics: New Progresses and Perspectives.

    Science.gov (United States)

    Zhan, Xianquan; Wang, Xiaowei; Cheng, Tingting

    2016-01-01

    Pituitary adenoma (PA) is a common intracranial neoplasm that impacts on human health through interfering hypothalamus-pituitary-target organ axis systems. The development of proteomics gives great promises in the clarification of molecular mechanisms of a PA and discovery of effective biomarkers for prediction, prevention, early-stage diagnosis, and treatment for a PA. A great progress in the field of PA proteomics has been made in the past 10 years, including (i) the use of laser-capture microdissection, (ii) proteomics analyses of functional PAs (such as prolactinoma), invasive and non-invasive non-functional pituitary adenomas (NFPAs), protein post-translational modifications such as phosphorylation and tyrosine nitration, NFPA heterogeneity, and hormone isoforms, (iii) the use of protein antibody array, (iv) serum proteomics and peptidomics, (v) the integration of proteomics and other omics data, and (vi) the proposal of multi-parameter systematic strategy for a PA. This review will summarize these progresses of proteomics in PAs, point out the existing drawbacks, propose the future research directions, and address the clinical relevance of PA proteomics data, in order to achieve our long-term goal that is use of proteomics to clarify molecular mechanisms, construct molecular networks, and discover effective biomarkers. PMID:27303365

  12. Human pituitary adenoma proteomics: new progresses and perspectives

    Directory of Open Access Journals (Sweden)

    Xianquan eZhan

    2016-05-01

    Full Text Available Pituitary adenoma (PA is a commonly intracranial neoplasm that impacts on human health through interfering hypothalamus-pituitary-target organ axis systems. The development of proteomics gives great promises in clarification of molecular mechanisms of a pituitary adenoma and discovery of effective biomarkers for prediction, prevention, early-stage diagnosis and treatment of a PA. A great progress in the field of PA proteomics has been made in the past ten years, including (i the use of laser capture microdissection, (ii proteomics analyses of functional PAs (FPAs, such as prolactinoma, invasive and noninvasive nonfunctional PAs (NFPAs, protein post-translational modifications (PTMs including phosphorylation and tyrosine nitration, NFPA heterogeneity, and hormone isoforms, (iii the use of protein antibody array, (iv serum proteomics and peptidomics, (v integration of proteomics and other omics data, and (vi proposal of multi-parameter systematic strategy for a PA. This review will summarize those progresses of proteomics in PAs, point out the existing drawbacks, propose the future research directions, and address the clinical relevance of PA proteomics data, in order to achieve our long-term goal that is use of proteomics to clarify molecular mechanisms, construct molecular networks, and discover effective biomarkers.

  13. Environmental Microbial Community Proteomics: Status, Challenges and Perspectives.

    Science.gov (United States)

    Wang, Da-Zhi; Kong, Ling-Fen; Li, Yuan-Yuan; Xie, Zhang-Xian

    2016-01-01

    Microbial community proteomics, also termed metaproteomics, is an emerging field within the area of microbiology, which studies the entire protein complement recovered directly from a complex environmental microbial community at a given point in time. Although it is still in its infancy, microbial community proteomics has shown its powerful potential in exploring microbial diversity, metabolic potential, ecological function and microbe-environment interactions. In this paper, we review recent advances achieved in microbial community proteomics conducted in diverse environments, such as marine and freshwater, sediment and soil, activated sludge, acid mine drainage biofilms and symbiotic communities. The challenges facing microbial community proteomics are also discussed, and we believe that microbial community proteomics will greatly enhance our understanding of the microbial world and its interactions with the environment. PMID:27527164

  14. Environmental Microbial Community Proteomics: Status, Challenges and Perspectives

    Science.gov (United States)

    Wang, Da-Zhi; Kong, Ling-Fen; Li, Yuan-Yuan; Xie, Zhang-Xian

    2016-01-01

    Microbial community proteomics, also termed metaproteomics, is an emerging field within the area of microbiology, which studies the entire protein complement recovered directly from a complex environmental microbial community at a given point in time. Although it is still in its infancy, microbial community proteomics has shown its powerful potential in exploring microbial diversity, metabolic potential, ecological function and microbe-environment interactions. In this paper, we review recent advances achieved in microbial community proteomics conducted in diverse environments, such as marine and freshwater, sediment and soil, activated sludge, acid mine drainage biofilms and symbiotic communities. The challenges facing microbial community proteomics are also discussed, and we believe that microbial community proteomics will greatly enhance our understanding of the microbial world and its interactions with the environment. PMID:27527164

  15. The cell-surface proteome of cultured adipose stromal cells.

    Science.gov (United States)

    Donnenberg, Albert D; Meyer, E Michael; Rubin, J Peter; Donnenberg, Vera S

    2015-07-01

    In this technical note we describe a method to evaluate the cell surface proteome of human primary cell cultures and cell lines. The method utilizes the BD Biosciences lyoplate, a system covering 242 surface proteins, glycoproteins, and glycosphingolipids plus relevant isotype controls, automated plate-based flow cytometry, conventional file-level analysis and unsupervised K-means clustering of markers on the basis of percent of positive events and mean fluorescence intensity of positive and total clean events. As an example, we determined the cell surface proteome of cultured adipose stromal cells (ASC) derived from 5 independent clinical isolates. Between-sample agreement of very strongly expressed (n = 32) and strongly expressed (n =16) markers was excellent, constituting a reliable profile for ASC identification and determination of functional properties. Known mesenchymal markers (CD29, CD44, CD73, CD90, CD105) were among the identified strongly expressed determinants. Among other strongly expressed markers are several that are potentially immunomodulatory including three proteins that protect from complement mediated effects (CD46, CD55, and CD59), two that regulate apoptosis (CD77 and CD95) and several with ectoenzymatic (CD10, CD26, CD13, CD73, and CD143) or receptor tyrosine kinase (CD140b (PDGFR), CD340 (Her-2), EGFR) activity, suggesting mechanisms for the anti-inflammatory and tissue remodeling properties of ASC. Because variables are standardized for K-means clustering, results generated using this methodology should be comparable between instrumentation platforms. It is widely generalizable to human primary explant cultures and cells lines and will prove useful to determine how cell passage, culture interventions, and gene expression and silencing affect the cell-surface proteome. PMID:25929697

  16. Drafting the proteome landscape of myeloid-derived suppressor cells.

    Science.gov (United States)

    Gato, María; Blanco-Luquin, Idoia; Zudaire, Maribel; de Morentin, Xabier Martínez; Perez-Valderrama, Estela; Zabaleta, Aintzane; Kochan, Grazyna; Escors, David; Fernandez-Irigoyen, Joaquín; Santamaría, Enrique

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently suppress T-cell responses. They regulate immune responses and the population significantly increases in the tumor microenvironment of patients with glioma and other malignant tumors. For their study, MDSCs are usually isolated from the spleen or directly of tumors from a large number of tumor-bearing mice although promising ex vivo differentiated MDSC production systems have been recently developed. During the last years, proteomics has emerged as a powerful approach to analyze MDSCs proteomes using shotgun-based mass spectrometry (MS), providing functional information about cellular homeostasis and metabolic state at a global level. Here, we will revise recent proteome profiling studies performed in MDSCs from different origins. Moreover, we will perform an integrative functional analysis of the protein compilation derived from these large-scale proteomic studies in order to obtain a comprehensive view of MDSCs biology. Finally, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and post-translational modifications (PTMs) during the differentiation process of MDSCs that will greatly boost the identification of novel MDSC-specific therapeutic targets to apply in cancer immunotherapy. PMID:26403437

  17. A proteomics perspective: from animal welfare to food safety.

    Science.gov (United States)

    Bassols, Anna; Turk, Romana; Roncada, Paola

    2014-03-01

    A fundamental issue of farm animal welfare is to keep animals clinically healthy, without disease or stress, particularly in intensive breeding, in order to produce safe and quality food. This issue is highly relevant for the food industry worldwide as they are directly linked to public health and welfare. The aim of this review is to explore how proteomics can assess and improve the knowledge useful for the strategic management of products of animal origin. Useful indications are provided about the latest proteomics tools for the development of novel biotechnologies serving the public health. The multivariate proteomics approach provides the bases for the discovery of biomarkers useful to investigate adaptation syndromes and oxidative stress. These two responses represent the milestones for the study of animal welfare. Moreover their implementation in the characterization and standardization of raw materials, process development, and quality and safety control of the final product of animal origin represents the current frontier in official surveillance and tests development. PMID:24555902

  18. WallProtDB, a database resource for plant cell wall proteomics

    OpenAIRE

    San Clemente, Hélène; Jamet, Elisabeth

    2015-01-01

    Background During the last fifteen years, cell wall proteomics has become a major research field with the publication of more than 50 articles describing plant cell wall proteomes. The WallProtDB database has been designed as a tool to facilitate the inventory, the interpretation of cell wall proteomics data and the comparisons between cell wall proteomes. Results WallProtDB (http://www.polebio.lrsv.ups-tlse.fr/WallProtDB/) presently contains 2170 proteins and ESTs identified experimentally i...

  19. Advancing cell biology through proteomics in space and time (PROSPECTS)

    DEFF Research Database (Denmark)

    Lamond, A.I.; Uhlen, M.; Horning, S.;

    2012-01-01

    a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU...

  20. The cell surface proteome of Staphylococcus aureus

    NARCIS (Netherlands)

    Dreisbach, Annette; van Dijl, Jan Maarten; Buist, Girbe

    2011-01-01

    The Gram-positive bacterium Staphylococcus aureus is a wide spread opportunistic pathogen that can cause a range of life-threatening diseases. To obtain a better understanding of the global mechanisms for pathogenesis and to identify novel targets for therapeutic interventions, the S. aureus proteom

  1. Unique proteomic signatures distinguish macrophages and dendritic cells.

    Directory of Open Access Journals (Sweden)

    Lev Becker

    Full Text Available Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.

  2. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  3. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Science.gov (United States)

    Niehage, Christian; Karbanová, Jana; Steenblock, Charlotte; Corbeil, Denis; Hoflack, Bernard

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2%) or high (10%) serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD) markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention. PMID:27490675

  4. Proteomic Analysis of Human Blastocoel Fluid and Blastocyst Cells

    DEFF Research Database (Denmark)

    Linnert Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen;

    cells of the blastocyst are exposed. The ICM is the starting point for the development of undifferentiated human embryonic stem cells (hESCs), which posses the potential to develop into any cell type present in the adult human body [1,2]. This ability makes hESCs a potential source of cells for...... blastocoel fluid (1-8 nanoliters per blastocyst), has hampered an in-depth study of the human blastocyst proteome. However, recent developments in mass spectrometry-based proteomic techniques allow the identification and characterization of thousands of proteins from low microgram levels of protein extracted......The human blastocyst consists of 100-200 cells that are organized in an outer layer of differentiated trophectoderm (TE) cells lining the blastocyst cavity into which the undifferentiated inner cell mass (ICM) protrudes. The cavity of the blastocyst is filled with blastocoel fluid to which all the...

  5. Proteomics of Important Food Crops in the Asia Oceania Region: Current Status and Future Perspectives

    KAUST Repository

    Chakraborty, Subhra

    2015-06-02

    In the rapidly growing economies of Asia and Oceania, food security has become a primary concern. With the rising population, growing more food at affordable prices is becoming even more important. In addition, the predicted climate change will lead to drastic changes in global surface temperature and changes in rainfall patterns that in turn would pose a serious threat to plant vegetation worldwide. As a result, understanding how plants will survive in a changing climate will be increasingly important. Such challenges require integrated approaches to increase agricultural production and cope with environmental threats. Proteomics can play a role in unravel the underlying mechanisms for food production to address the growing demand for food. In this review, the current status of food crop proteomics is discussed, especially in regards to the Asia and Oceania regions. Furthermore, the future perspective in relation to proteomic techniques for the important food crops is highlighted.

  6. Proteomics of CDK inhibition in cancer cells

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Hana; Skalníková, Helena; Halada, Petr; Strnad, M.; Hajdúch, M.

    Olomouc: -, 2007, s. 1-1. [Symposium and Workshop on Molecular Pathology /3./. Olomouc (CZ), 04.05.2007-05.05.2007] R&D Projects: GA ČR GA301/05/0418; GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : cyclin-dependent kinase inhibitors * cancer * proteomics Subject RIV: EB - Genetics ; Molecular Biology

  7. Proteomic techniques for characterisation of mesenchymal stem cell secretome.

    Czech Academy of Sciences Publication Activity Database

    Kupcová Skalníková, Helena

    2013-01-01

    Roč. 95, č. 12 (2013), s. 2196-2211. ISSN 0300-9084 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR TA01011466 Institutional support: RVO:67985904 Keywords : mesenchymal stem cells * secretome * exosome * conditioned medium * proteomics Subject RIV: CE - Biochemistry Impact factor: 3.123, year: 2013

  8. The time is right: proteome biology of stem cells.

    NARCIS (Netherlands)

    Whetton, A.D.; Williamson, A.J.K.; Krijgsveld, J.; Lee, B.H.; Lemischka, I.; Oh, S.; Pera, M.; Mummery, C.L.; Heck, A.J.R.

    2008-01-01

    In stem cell biology, there is a growing need for advanced technologies that may help to unravel the molecular mechanisms of self-renewal and differentiation. Proteomics, the comprehensive analysis of proteins, is such an emerging technique. To facilitate interactions between specialists in proteomi

  9. Proteomic Applications in the Study of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Jesús Mateos

    2014-02-01

    Full Text Available Mesenchymal stem cells (MSCs are undifferentiated cells with an unlimited capacity for self-renewal and able to differentiate towards specific lineages under appropriate conditions. MSCs are, a priori, a good target for cell therapy and clinical trials as an alternative to embryonic stem cells, avoiding ethical problems and the chance for malignant transformation in the host. However, regarding MSCs, several biological implications must be solved before their application in cell therapy, such as safe ex vivo expansion and manipulation to obtain an extensive cell quantity amplification number for use in the host without risk accumulation of genetic and epigenetic abnormalities. Cell surface markers for direct characterization of MSCs remain unknown, and the precise molecular mechanisms whereby growth factors stimulate their differentiation are still missing. In the last decade, quantitative proteomics has emerged as a promising set of techniques to address these questions, the answers to which will determine whether MSCs retain their potential for use in cell therapy. Proteomics provides tools to globally analyze cellular activity at the protein level. This proteomic profiling allows the elucidation of connections between broad cellular pathways and molecules that were previously impossible to determine using only traditional biochemical analysis. However; thus far, the results obtained must be orthogonally validated with other approaches. This review will focus on how these techniques have been applied in the evaluation of MSCs for their future applications in safe therapies.

  10. Learning robust cell signalling models from high throughput proteomic data

    OpenAIRE

    Koch, Mitchell; Broom, Bradley M.; Subramanian, Devika

    2009-01-01

    We propose a framework for learning robust Bayesian network models of cell signalling from high-throughput proteomic data. We show that model averaging using Bayesian bootstrap resampling generates more robust structures than procedures that learn structures using all of the data. We also develop an algorithm for ranking the importance of network features using bootstrap resample data. We apply our algorithms to derive the T-cell signalling network from the flow cytometry data of Sachs et al....

  11. Proteome analysis of bronchoalveolar lavage in pulmonary langerhans cell histiocytosis

    OpenAIRE

    2011-01-01

    Background Pulmonary Langerhans-cell histiocytosis (PLCH) is a rare interstitial lung disease characterized by clusters of Langerhans cells, organized in granulomas, in the walls of distal bronchioles. It is a diffuse lung disease related to tobacco smoking but otherwise of unknown etiopathogenesis. Methods In this study we used a proteomic approach to analyze BAL protein composition of patients with PLCH and of healthy smoker and non-smoker controls to obtain insights into the pathogenetic m...

  12. Global Proteome Analysis of the NCI-60 Cell Line Panel

    Directory of Open Access Journals (Sweden)

    Amin Moghaddas Gholami

    2013-08-01

    Full Text Available The NCI-60 cell line collection is a very widely used panel for the study of cellular mechanisms of cancer in general and in vitro drug action in particular. It is a model system for the tissue types and genetic diversity of human cancers and has been extensively molecularly characterized. Here, we present a quantitative proteome and kinome profile of the NCI-60 panel covering, in total, 10,350 proteins (including 375 protein kinases and including a core cancer proteome of 5,578 proteins that were consistently quantified across all tissue types. Bioinformatic analysis revealed strong cell line clusters according to tissue type and disclosed hundreds of differentially regulated proteins representing potential biomarkers for numerous tumor properties. Integration with public transcriptome data showed considerable similarity between mRNA and protein expression. Modeling of proteome and drug-response profiles for 108 FDA-approved drugs identified known and potential protein markers for drug sensitivity and resistance. To enable community access to this unique resource, we incorporated it into a public database for comparative and integrative analysis (http://wzw.tum.de/proteomics/nci60.

  13. Differential proteome analysis of chikungunya virus infection on host cells.

    Directory of Open Access Journals (Sweden)

    Christina Li-Ping Thio

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach. METHODOLOGY AND PRINCIPAL FINDINGS: The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE. Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP and cell cycle regulation. CONCLUSION/SIGNIFICANCE: This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1 regulation (in favour of virus survival, replication and transmission. While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.

  14. What Is Cancer Proteomics?

    Science.gov (United States)

    ... What is Proteomics? Video Tutorial What is Cancer Proteomics? Print This Page The term "proteome" refers to ... that a cell or organism undergoes. The term "proteomics" is a large-scale comprehensive study of a ...

  15. Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

    OpenAIRE

    Sood, Anup; Miller, Alexandra M.; Brogi, Edi; Sui, Yunxia; Armenia, Joshua; McDonough, Elizabeth; Santamaria-Pang, Alberto; Carlin, Sean; Stamper, Aleksandra; Campos, Carl; Pang, Zhengyu; Li, Qing; Port, Elisa; Graeber, Thomas G.; Schultz, Nikolaus

    2016-01-01

    The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised cluster...

  16. Mass spectrometry based proteomics for absolute quantification of proteins from tumor cells

    OpenAIRE

    Wang, Hong; Hanash, Sam

    2015-01-01

    In-depth quantitative profiling of the proteome and sub-proteomes of tumor cells has relevance to tumor classification, the development of novel therapeutics, and of prognostic and predictive markers and to disease monitoring. In particular the tumor cell surface represents a highly relevant compartment for the development of targeted therapeutics and immunotherapy. We have developed a proteomic platform to profile tumor cells that encompasses enrichment of surface membrane proteins, intact p...

  17. Mass spectrometry based proteomics in cell biology and signaling research

    International Nuclear Information System (INIS)

    Full text: Proteomics is one of the most powerful post-genomics technologies. Recently accomplishments include large scale protein-protein interaction mapping, large scale mapping of phosphorylation sites and the cloning of key signaling molecules. In this talk, current state of the art of the technology will be reviewed. Applications of proteomics to the mapping of multiprotein complexes will be illustrated with recent work on the spliceosome and the nucleolus. More than 300 proteins have been mapped to each of these complexes. Quantitative techniques are becoming more and more essential in proteomics. They are usually performed by the incorporation of stable isotopes - a light form in cell state 'A' and a heavy form in cell state 'E' - and subsequent comparison of mass spectrometric peak heights. A new technique called, SILAC for Stable isotope Incorporation by Amino acids in Cell culture, has been applied to studying cell differentiation and mapping secreted proteins from adipocytes. A number of known and novel proteins important in adipocyte differentiation have been identified by this technique. Some of these proved to be upregulated at the 1 mRNA level, too, whereas others appear to be regulated post-translationally. We have also applied the SILAC method to protein-protein interaction mapping. For example, we compared immunoprecipitates from stimulated and non-stimulated cells to find binding partners recruited to the bait due to the stimulus. Several novel substrates in the EGF pathway were found in this way. An important application of proteomics in the signaling field is the mapping of post-translational modifications. In particular, there are a number of techniques for phosphotyrosine phosphorylation mapping which have proven very useful. Making use of the mass deficiency of the phosphogroup, 'parent ion scans' con be performed, which selectively reveal phosphotyrosine peptides from complex peptides mixtures. This technique has been used to clone several

  18. Application of proteomics in non-small-cell lung cancer.

    Science.gov (United States)

    Cho, William C S

    2016-01-01

    Non-small-cell lung cancer (NSCLC) is a heterogeneous disease with diverse pathological features. Clinical proteomics allows the discovery of molecular markers and new therapeutic targets for this most prevalent type of lung cancer. Some of them may be used to detect early lung cancer, while others may serve as predictive markers of resistance to different therapies. Therapeutic targets and prognostic markers in NSCLC have also been discovered. These proteomics biomarkers may help to pair the individual NSCLC patient with the best treatment option. Despite the fact that implementation of these biomarkers in the clinic appears to be scarce, the recently launched Precision Medicine Initiative may encourage their translation into clinical practice. PMID:26577456

  19. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    Science.gov (United States)

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future. PMID:25211708

  20. Cerebrospinal fluid proteomics and protein biomarkers in frontotemporal lobar degeneration: Current status and future perspectives.

    Science.gov (United States)

    Oeckl, Patrick; Steinacker, Petra; Feneberg, Emily; Otto, Markus

    2015-07-01

    Frontotemporal lobar degeneration (FTLD) comprises a spectrum of rare neurodegenerative diseases with an estimated prevalence of 15-22 cases per 100,000 persons including the behavioral variant of frontotemporal dementia (bvFTD), progressive non-fluent aphasia (PNFA), semantic dementia (SD), FTD with motor neuron disease (FTD-MND), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS). The pathogenesis of the diseases is still unclear and clinical diagnosis of FTLD is hampered by overlapping symptoms within the FTLD subtypes and with other neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Intracellular protein aggregates in the brain are a major hallmark of FTLD and implicate alterations in protein metabolism or function in the disease's pathogenesis. Cerebrospinal fluid (CSF) which surrounds the brain can be used to study changes in neurodegenerative diseases and to identify disease-related mechanisms or neurochemical biomarkers for diagnosis. In the present review, we will give an overview of the current literature on proteomic studies in CSF of FTLD patients. Reports of targeted and unbiased proteomic approaches are included and the results are discussed in regard of their informative value about disease pathology and the suitability to be used as diagnostic biomarkers. Finally, we will give some future perspectives on CSF proteomics and a list of candidate biomarkers which might be interesting for validation in further studies. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology. PMID:25526887

  1. Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins.

    Science.gov (United States)

    Wang, Chenyuan; Liu, Yang; Chang, Cheng; Wu, Songfeng; Gao, Jie; Zhang, Yang; Chen, Yingjie; Zhong, Fan; Deng, Gaopi

    2016-01-01

    The object of this research was to report a draft proteome of human fallopian tube (hFT) comprises 5416 identified proteins, which could be considered as a physiological reference to complement Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome contains many hFT markers newly identified by mass spectrum. This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, vesicle and protein folding associated proteins showed obvious tendency to be higher abundance in hFT. The extraordinary high coverage of mesenchymal stem cells (MSCs)-associated proteins were identified in this hFT proteome, which highly supported that hFT should contain a plenty of MSCs. PMID:26759384

  2. Recent advances in atherosclerosis-based proteomics: new biomarkers and a future perspective.

    Science.gov (United States)

    Alvarez-Llamas, Gloria; de la Cuesta, Fernando; Barderas, Maria Eugenia G; Darde, Veronica; Padial, Luis R; Vivanco, Fernando

    2008-10-01

    Vascular proteomics is providing two main types of data: proteins that actively participate in vascular pathophysiological processes and novel protein candidates that can potentially serve as useful clinical biomarkers. Although both types of proteins can be identified by similar proteomic strategies and methods, it is important to clearly distinguish biomarkers from mediators of disease. A particular protein, or group of proteins, may participate in a pathogenic process but not serve as an effective biomarker. Alternatively, a useful biomarker may not mediate pathogenic pathways associated with disease (i.e., C-reactive protein). To date, there are no clear successful examples in which discovery proteomics has led to a novel useful clinical biomarker in cardiovascular diseases. Nevertheless, new sources of biomarkers are being explored (i.e., secretomes, circulating cells, exosomes and microparticles), an increasing number of novel proteins involved in atherogenesis are constantly described, and new technologies and analytical strategies (i.e., quantitative proteomics) are being developed to access low abundant proteins. Therefore, this presages a new era of discovery and a further step in the practical application to diagnosis, prognosis and early action by medical treatment of cardiovascular diseases. PMID:18937558

  3. A Proteomic Study of Human Merkel Cell Carcinoma.

    Science.gov (United States)

    Shao, Qiang; Byrum, Stephanie D; Moreland, Linley E; Mackintosh, Samuel G; Kannan, Aarthi; Lin, Zhenyu; Morgan, Michael; Stack, Brendan C; Cornelius, Lynn A; Tackett, Alan J; Gao, Ling

    2013-11-25

    Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin. The incidence has been quadrupled with a 5-year mortality rate of 46%, presently there is no cure for metastatic disease. Despite the contribution of Merkel cell polyomavirus, the molecular events of MCC carcinogenesis are poorly defined. To better understand MCC carcinogensis, we have performed the first quantitative proteomic comparison of formalin-fixed, paraffin-embedded (FFPE) MCC tissues using another neuroendocrine tumor (carcinoid tumor of the lung) as controls. Bioinformatic analysis of the proteomic data has revealed that MCCs carry distinct protein expression patterns. Further analysis of significantly over-expressed proteins suggested the involvement of MAPK, PI3K/Akt/mTOR, wnt, and apoptosis signaling pathways. Our previous study and that from others have shown mTOR activation in MCCs. Therefore, we have focused on two downstream molecules of the mTOR pathway, lactate dehydrogenase B (LDHB) and heterogeneous ribonucleoprotein F (hnRNPF). We confirm over-expression of LDHB and hnRNPF in two primary human MCC cell lines, 16 fresh tumors, and in the majority of 80 tissue microarray samples. Moreover, mTOR inhibition suppresses LDHB and hnRNPF expression in MCC cells. The results of the current study provide insight into MCC carcinogenesis and provide rationale for mTOR inhibition in pre-clinical studies. PMID:25284964

  4. Intriguing bronchoalveolar lavage proteome in a case of pulmonary langerhans cell histiocytosis

    OpenAIRE

    Ghafouri, Bijar; Persson, H Lennart; Tagesson, Christer

    2013-01-01

    Background: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease associated with tobacco smoke exposure. New insights into its pathogenesis and how it differs from that of chronic obstructive pulmonary disease (COPD) may be provided by proteomic studies on bronchoalveolar lavage fluid (BALF). Case Report: We present the BALF proteome in a biopsy-proven case of PLCH and compare it with typical proteomes of COPD and of the healthy lung. The BALF proteins were separ...

  5. Proteome of leukaemic cells after cisplatin treatment - comparison of three independent analytical techniques

    Czech Academy of Sciences Publication Activity Database

    Martinková, Jiřina; Skalníková, Helena; Hrabáková, Rita; Novák, Petr; Man, Petr; Pompach, Petr; Strohalm, Martin; Havlíček, Vladimír; Džubák, P.; Hajdúch, M.; Kovářová, Hana

    Budapest : Hungarian proteomic society, 2009, s. 56-56. ISBN 978-963-9319-99-8. [3rd Central and Eastern European Proteomics Conference. Budapešť (HU), 06.10.2009-09.10.2009] R&D Projects: GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50450515 Keywords : Proteomics * Cis-platin * Leukaemic cells Subject RIV: FK - Gynaecology, Childbirth

  6. Characterization of the Sclerotinia sclerotiorum cell wall proteome.

    Science.gov (United States)

    Liu, Longzhou; Free, Stephen J

    2016-08-01

    We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. PMID:26661933

  7. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

    DEFF Research Database (Denmark)

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian;

    2009-01-01

    Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network of...... after osteogenic differentiation. We also identified regulatory proteins, such as the transcription factors TP53 and Sp-1, associated with the differentiation process. Further studies will investigate the impact of identified regulatory proteins for cell proliferation and osteogenic differentiation in...... differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B...

  8. Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots

    Science.gov (United States)

    Zhu, Yingde; Li, Hui; Bhatti, Sarabjit; Zhou, Suping; Yang, Yong; Fish, Tara; Thannhauser, Theodore W

    2016-01-01

    Single-cell-type proteomics provides the capability to revealing the genomic and proteomics information at cell-level resolution. However, the methodology for this type of research has not been well-developed. This paper reports developing a workflow of laser capture microdissection (LCM) followed by gel-liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)-based proteomics analysis for the identification of proteomes contained in individual cell layers of tomato roots. Thin-sections (~10-μm thick, 10 sections per root tip) were prepared for root tips of tomato germinating seedlings. Epidermal and cortical cells (5000–7000 cells per tissue type) were isolated under a LCM microscope. Proteins were isolated and then separated by SDS–polyacrylamide gel electrophoresis followed by in-gel-tryptic digestion. The MS and MS/MS spectra generated using nanoLC-MS/MS analysis of the tryptic peptides were searched against ITAG2.4 tomato protein database to identify proteins contained in each single-cell-type sample. Based on the biological functions, proteins with proven functions in root hair development were identified in epidermal cells but not in the cortical cells. Several of these proteins were found in Al-treated roots only. The results demonstrated that the cell-type-specific proteome is relevant for tissue-specific functions in tomato roots. Increasing the coverage of proteomes and reducing the inevitable cross-contamination from adjacent cell layers, in both vertical and cross directions when cells are isolated from slides prepared using intact root tips, are the major challenges using the technology in proteomics analysis of plant roots. PMID:27280026

  9. Iron overload in human hepatoma cells - proteomic analysis

    Czech Academy of Sciences Publication Activity Database

    Petrák, J.; Myslivcová, D.; Man, Petr; Babušiak, M.; Vyoral, D.

    Lednice, 2005, s. 38-38. [Czech Proteomic Conference /2./. Lednice (CZ), 17.10.2005-20.10.2005] Institutional research plan: CEZ:AV0Z50200510 Keywords : iron * proteomic analysis Subject RIV: EE - Microbiology, Virology

  10. Proteomic assessment of a cell model of spinal muscular atrophy

    Directory of Open Access Journals (Sweden)

    Lee Kelvin H

    2011-03-01

    Full Text Available Abstract Background Deletion or mutation(s of the survival motor neuron 1 (SMN1 gene causes spinal muscular atrophy (SMA, a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease. Results When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8 in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament and motor neuron markers (Hb9, Islet-1, and ChAT. Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells, the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived. Conclusion SMN

  11. Comparative proteomic analysis on radioresistant nasopharyngeal carcinoma cell

    International Nuclear Information System (INIS)

    Objective: To discover radioresistance-associated proteins by performing comparative proteomic analysis on nasopharyngeal carcinoma cell lines. Methods: The total proteins were extracted from radioresistant human nasopharyngeal carcinoma cell line CNE-2R and its parental cell line CNE-2, respectively. These proteins were separated by high quality two-dimensional polyacrylamide gel electrophoresis (2-DE) and then the 2-DE profiles were screened for differentially expressed protein spots by the Image Master 5.0 software. Those spots were identified by a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Results: 32 significantly differentially expressed protein spots were screened in two different radiosensitivity cell lines and 11 proteins were identified by tandem mass spectrometry, among which 3 proteins were up-regulated in radioresistant human nasopharyngeal carcinoma cell line CNE-2R and the other 8 proteins were down-regulated. Conclusions: The differentially expressed proteins of nasopharyngeal carcinoma cells with different radiosensitivity were mainly involved in apoptosis regulation, DNA damage and repair, cell cycle regulation, RNA transcription, cell signaling, cytoskeleton formation and radiation stress responses. (authors)

  12. Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines

    OpenAIRE

    Paulo, Joao A.; Kadiyala, Vivek; Banks, Peter A; Conwell, Darwin L; Steen, Hanno

    2013-01-01

    The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immorta...

  13. Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

    OpenAIRE

    Min, Hophil; Han, Dohyun; Kim, Yikwon; Cho, Jee Yeon; Jin, Jonghwa; Kim, Youngsoo

    2014-01-01

    Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage ...

  14. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    OpenAIRE

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Ba...

  15. Biological Networks Underlying Abiotic Stress Tolerance in Temperate Crops—A Proteomic Perspective

    Directory of Open Access Journals (Sweden)

    Klára Kosová

    2015-09-01

    Full Text Available Abiotic stress factors, especially low temperatures, drought, and salinity, represent the major constraints limiting agricultural production in temperate climate. Under the conditions of global climate change, the risk of damaging effects of abiotic stresses on crop production increases. Plant stress response represents an active process aimed at an establishment of novel homeostasis under altered environmental conditions. Proteins play a crucial role in plant stress response since they are directly involved in shaping the final phenotype. In the review, results of proteomic studies focused on stress response of major crops grown in temperate climate including cereals: common wheat (Triticum aestivum, durum wheat (Triticum durum, barley (Hordeum vulgare, maize (Zea mays; leguminous plants: alfalfa (Medicago sativa, soybean (Glycine max, common bean (Phaseolus vulgaris, pea (Pisum sativum; oilseed rape (Brassica napus; potato (Solanum tuberosum; tobacco (Nicotiana tabaccum; tomato (Lycopersicon esculentum; and others, to a wide range of abiotic stresses (cold, drought, salinity, heat, imbalances in mineral nutrition and heavy metals are summarized. The dynamics of changes in various protein functional groups including signaling and regulatory proteins, transcription factors, proteins involved in protein metabolism, amino acid metabolism, metabolism of several stress-related compounds, proteins with chaperone and protective functions as well as structural proteins (cell wall components, cytoskeleton are briefly overviewed. Attention is paid to the differences found between differentially tolerant genotypes. In addition, proteomic studies aimed at proteomic investigation of multiple stress factors are discussed. In conclusion, contribution of proteomic studies to understanding the complexity of crop response to abiotic stresses as well as possibilities to identify and utilize protein markers in crop breeding processes are discussed.

  16. Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

    Directory of Open Access Journals (Sweden)

    Mafumi Abiko

    Full Text Available In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

  17. Proteomic Studies of Cholangiocarcinoma and Hepatocellular Carcinoma Cell Secretomes

    Directory of Open Access Journals (Sweden)

    Chantragan Srisomsap

    2010-01-01

    Full Text Available Cholangiocarcinoma (CCA and hepatocellular carcinoma (HCC occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1 and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2, laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma.

  18. Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomics studies

    OpenAIRE

    Zhang, Ying; Bottinelli, Dario; Lisacek, Frédérique; Luban, Jeremy; De Castillia, Caterina Strambio; Varesio, Emmanuel; Hopfgartner, Gérard

    2015-01-01

    Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize mass spectrometry coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilisation and denaturation methods were ...

  19. Proteomic study of cancer cells response to anthracycline anticancer treatment

    Czech Academy of Sciences Publication Activity Database

    Tylečková, Jiřina; Hrabáková, Rita; Mairychová, Kateřina; Halada, Petr; Radová, L.; Džubák, P.; Hajduch, M.; Kovářová, Hana

    Berlin : Freie Universität Berlin, 2011. s. 225-225. [Proteomic Forum 2011. 3.4.2011-7.4.2011, Berlin] R&D Projects: GA MŠk LC07017; GA MŠk MSM6198959216 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : anticancer research * anthracycline * proteomics Subject RIV: CE - Biochemistry http://www.proteomic-forum.de/images/stories/pics/proteomic%20forum%202011%20-%20abstract%20book.pdf

  20. Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

    Science.gov (United States)

    Sood, Anup; Miller, Alexandra M.; Brogi, Edi; Sui, Yunxia; Armenia, Joshua; McDonough, Elizabeth; Santamaria-Pang, Alberto; Carlin, Sean; Stamper, Aleksandra; Campos, Carl; Pang, Zhengyu; Li, Qing; Port, Elisa; Graeber, Thomas G.; Schultz, Nikolaus; Ginty, Fiona; Larson, Steven M.; Mellinghoff, Ingo K.

    2016-01-01

    The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes. PMID:27182557

  1. Optimized method for identification of the proteomes secreted by cardiac cells

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Van Eyk, J.E.

    2013-01-01

    Roč. 1005, č. 1005 (2013), s. 225-235. ISSN 1940-6029 Institutional support : RVO:68081715 Keywords : cardiac cells * secreted proteins * proteomic technology Subject RIV: CB - Analytical Chemistry, Separation

  2. Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae).

    Science.gov (United States)

    Wang, Sheng-Bing; Hu, Qiang; Sommerfeld, Milton; Chen, Feng

    2004-03-01

    The green microalga Haematococcus pluvialis can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, and undergo profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stresses. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with peptide mass fingerprinting (PMF) and sequence-database analysis. In total, 163 protein bands were analyzed, which resulted in positive identification of 81 protein orthologues. The highly complex and dynamic composition of CWPs is manifested by the fact that the majority of identified CWPs are differentially expressed at specific stages of the cell cycle along with a number of common wall-associated 'housekeeping' proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation, in contrast to earlier observations that cellulose was exclusively present in the secondary wall of the organism. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation and/or assisting in cell expansion. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and the specific protein markers obtained from this study provide a framework for future characterization of the expression and physiological functions of the proteins involved in the biogenesis and modifications in the cell wall of Haematococcus and related organisms. PMID:14997492

  3. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  4. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    International Nuclear Information System (INIS)

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs

  5. Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

    Science.gov (United States)

    Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

    2016-01-01

    The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

  6. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

    Directory of Open Access Journals (Sweden)

    Samuel T. Mindaye

    2015-12-01

    Full Text Available Bone-marrow derived mesenchymal stromal cells (BMSCs have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5,6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  7. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  8. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    Directory of Open Access Journals (Sweden)

    Ilona Turek

    2015-09-01

    Full Text Available Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP, AtPNP-A (At2g18660 were assessed using quantitative proteomics employing tandem mass tag (TMT labeling and tandem mass spectrometry (LC–MS/MS. In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014 661 and have been deposited to the ProteomeXchange with identifier PXD001386.

  9. Proteomic differences in recombinant CHO cells producing two similar antibody fragments.

    Science.gov (United States)

    Sommeregger, Wolfgang; Mayrhofer, Patrick; Steinfellner, Willibald; Reinhart, David; Henry, Michael; Clynes, Martin; Meleady, Paula; Kunert, Renate

    2016-09-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. "Omics" studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label-free LC-MS proteomic analyses to investigate product-specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single-chain Fv-Fc homodimeric antibody fragments (scFv-Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase-mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label-free proteomic analysis. LC-MS-MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902-1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26913574

  10. Proteomics of Neural Stem Cell Differentiation: from Animal Model to Human

    Czech Academy of Sciences Publication Activity Database

    Mairychová, Kateřina; Skalníková, Helena; Tylečková, Jiřina; Halada, Petr; Marsala, M.; Kovářová, Hana

    Berlin : Freie Universität Berlin, 2011. s. 211-211. [Proteomic Forum 2011. 03.04.2011-07.04.2011, Berlin] R&D Projects: GA MŠk 1M0538; GA MŠk(CZ) ME10044 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : stem cell s * differentiation * proteomics Subject RIV: FH - Neurology

  11. Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis.

    OpenAIRE

    Borderies, Gisèle; Jamet, Elisabeth; Lafitte, Claude; Rossignol, Michel; Jauneau, Alain; Boudart, Georges; Monsarrat, Bernard; Esquerré-Tugayé, Marie-Thérèse; Boudet, Alain; Pont-Lezica, Rafael

    2003-01-01

    The complete sequencing of the Arabidopsis thaliana genome allows the use of the recently developed mass spectrometry techniques to identify the cell wall proteins (CWPs). Most proteomic approaches depend on the quality of sample preparation. Extraction of CWPs is particularly complex since the proteins may be free in the apoplast or are embedded in a polysaccharide matrix where they are retained by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions, or cross-linked...

  12. Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation.

    Science.gov (United States)

    Wilson, Marieangela C; Trakarnsanga, Kongtana; Heesom, Kate J; Cogan, Nicola; Green, Carole; Toye, Ashley M; Parsons, Steve F; Anstee, David J; Frayne, Jan

    2016-06-01

    Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2- and 100-fold following maturation. However, ∼5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin. PMID:27006477

  13. Functional Proteomics Screen Enables Enrichment of Distinct Cell Types from Human Pancreatic Islets

    OpenAIRE

    Revital Sharivkin; Walker, Michael D.; Yoav Soen

    2015-01-01

    The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates sp...

  14. Proteome alteration induced by hTERT transfection of human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Riou Jean-François

    2008-04-01

    Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

  15. Immunoscreening of the extracellular proteome of colorectal cancer cells

    International Nuclear Information System (INIS)

    The release of proteins from tumors can trigger an immune response in cancer patients involving T lymphocytes and B lymphocytes, which results in the generation of antibodies to tumor-derived proteins. Many studies aim to use humoral immune responses, namely autoantibody profiles, directly, as clinical biomarkers. Alternatively, the antibody immune response as an amplification system for tumor associated alterations may be used to indicate putative protein biomarkers with high sensitivity. Aiming at the latter approach we here have implemented an autoantibody profiling strategy which particularly focuses on proteins released by tumor cells in vitro: the so-called secretome. For immunoscreening, the extracellular proteome of five colorectal cancer cell lines was resolved on 2D gels, immobilized on PVDF membranes and used for serological screening with individual sera from 21 colorectal cancer patients and 24 healthy controls. All of the signals from each blot were assigned to a master map, and autoantigen candidates were defined based of the pattern of immunoreactivities. The corresponding proteins were isolated from preparative gels, identified by MALDI-MS and/or by nano-HPLC/ESI-MS/MS and exemplarily confirmed by duplex Western blotting combining the human serum samples with antibodies directed against the protein(s) of interest. From 281 secretome proteins stained with autoantibodies in total we first defined the 'background patterns' of frequently immunoreactive extracellular proteins in healthy and diseased people. An assignment of these proteins, among them many nominally intracellular proteins, to the subset of exosomal proteins within the secretomes revealed a large overlap. On this basis we defined and consequently confirmed novel biomarker candidates such as the extreme C-terminus of the extracellular matrix protein agrin within the set of cancer-enriched immunorectivities. Our findings suggest, first, that autoantibody responses may be due, in

  16. Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

    Directory of Open Access Journals (Sweden)

    Heiner Falkenberg

    2013-01-01

    Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

  17. Biochemistry, proteomics, and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    DEFF Research Database (Denmark)

    Havelund, Jesper; Thelen, Jay J.; Møller, Ian Max

    2013-01-01

    functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of...

  18. Proteomics in Cell Culture: From Genomics to Combined ‘Omics for Cell Line Engineering and Bioprocess Development

    DEFF Research Database (Denmark)

    Heffner, Kelley; Kaas, Christian Schrøder; Kumar, Amit;

    2015-01-01

    in media development and cell line engineering to improve growth or productivity, delay the onset of apoptosis, or utilize nutrients efficiently. Mass-spectrometry based and other proteomics methods can provide for the detection of thousands of proteins from cell culture and bioinformatics analysis...... protein production has increased significantly because proteomics can track changes in protein levels for different cell lines over time, which can be advantageous for bioprocess development and optimization. Specifically, the identification of proteins that affect cell culture processes can aid efforts...

  19. Designing biomedical proteomics experiments: state-of-the-art and future perspectives.

    Science.gov (United States)

    Maes, Evelyne; Kelchtermans, Pieter; Bittremieux, Wout; De Grave, Kurt; Degroeve, Sven; Hooyberghs, Jef; Mertens, Inge; Baggerman, Geert; Ramon, Jan; Laukens, Kris; Martens, Lennart; Valkenborg, Dirk

    2016-05-01

    With the current expanded technical capabilities to perform mass spectrometry-based biomedical proteomics experiments, an improved focus on the design of experiments is crucial. As it is clear that ignoring the importance of a good design leads to an unprecedented rate of false discoveries which would poison our results, more and more tools are developed to help researchers designing proteomic experiments. In this review, we apply statistical thinking to go through the entire proteomics workflow for biomarker discovery and validation and relate the considerations that should be made at the level of hypothesis building, technology selection, experimental design and the optimization of the experimental parameters. PMID:27031651

  20. The Transcriptomics to Proteomics of Hair Cell Regeneration: Looking for a Hair Cell in a Haystack

    Directory of Open Access Journals (Sweden)

    Michael E. Smith

    2013-07-01

    Full Text Available Mature mammals exhibit very limited capacity for regeneration of auditory hair cells, while all non-mammalian vertebrates examined can regenerate them. In an effort to find therapeutic targets for deafness and balance disorders, scientists have examined gene expression patterns in auditory tissues under different developmental and experimental conditions. Microarray technology has allowed the large-scale study of gene expression profiles (transcriptomics at whole-genome levels, but since mRNA expression does not necessarily correlate with protein expression, other methods, such as microRNA analysis and proteomics, are needed to better understand the process of hair cell regeneration. These technologies and some of the results of them are discussed in this review. Although there is a considerable amount of variability found between studies owing to different species, tissues and treatments, there is some concordance between cellular pathways important for hair cell regeneration. Since gene expression and proteomics data is now commonly submitted to centralized online databases, meta-analyses of these data may provide a better picture of pathways that are common to the process of hair cell regeneration and lead to potential therapeutics. Indeed, some of the proteins found to be regulated in the inner ear of animal models (e.g., IGF-1 have now gone through human clinical trials.

  1. Human Cancer Classification: A Systems Biology- Based Model Integrating Morphology, Cancer Stem Cells, Proteomics, and Genomics

    OpenAIRE

    Halliday A Idikio

    2011-01-01

    Human cancer classification is currently based on the idea of cell of origin, light and electron microscopic attributes of the cancer. What is not yet integrated into cancer classification are the functional attributes of these cancer cells. Recent innovative techniques in biology have provided a wealth of information on the genomic, transcriptomic and proteomic changes in cancer cells. The emergence of the concept of cancer stem cells needs to be included in a classification model to capture...

  2. Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells.

    Directory of Open Access Journals (Sweden)

    Lucie Lorkova

    Full Text Available Mantle cell lymphoma (MCL is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino. We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine and to an inhibitor of Bruton tyrosine kinase (BTK ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib or remained unaffacted (cisplatin, bendamustine. The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib, but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.

  3. Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell

    DEFF Research Database (Denmark)

    Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris;

    2008-01-01

    Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much...... identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human...

  4. Open source libraries and frameworks for mass spectrometry based proteomics: A developer's perspective

    OpenAIRE

    Perez-Riverol, Yasset; Wang, Rui; Hermjakob, Henning; Müller, Markus; Müller, Markus; Vesada, Vladimir; Vizcaíno, Juan Antonio; Vizcaíno, Juan Antonio

    2014-01-01

    Data processing, management and visualization are central and critical components of a state of the art high-throughput mass spectrometry (MS)-based proteomics experiment, and are often some of the most time-consuming steps, especially for labs without much bioinformatics support. The growing interest in the field of proteomics has triggered an increase in the development of new software libraries, including freely available and open-source software. From database search analysis to post-proc...

  5. Proteomic profiling of acrolein adducts in human lung epithelial cells

    OpenAIRE

    Spiess, Page C; Deng, Bin; Hondal, Robert J.; Matthews, Dwight E.; van der Vliet, Albert

    2011-01-01

    Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway ep...

  6. Integration of genomics, proteomics, and imaging for cardiac stem cell therapy

    International Nuclear Information System (INIS)

    Cardiac stem cell therapy is beginning to mature as a valid treatment for heart disease. As more clinical trials utilizing stem cells emerge, it is imperative to establish the mechanisms by which stem cells confer benefit in cardiac diseases. In this paper, we review three methods - molecular cellular imaging, gene expression profiling, and proteomic analysis - that can be integrated to provide further insights into the role of this emerging therapy. (orig.)

  7. Effects of retinoic acid isomers on proteomic pattern in human breast cancer MCF-7 cell line

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Benkovská, Dagmar; Macejová, D.; Bialešová, L.; Bobálová, Janette; Brtko, J.

    2013-01-01

    Roč. 47, č. 4 (2013), s. 205-209. ISSN 1210-0668 R&D Projects: GA MŠk(CZ) 7AMB12SK151 Institutional support: RVO:68081715 Keywords : retinoic acid isomers * retinoid * breast cancer * malignant cells * proteomic analysis Subject RIV: CB - Analytical Chemistry, Separation

  8. Specificity of secreted proteomes from cardiac stem cells and neonatal myocytes

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Chimenti, I.; Marban, E.; Van Eyk, J.

    2009-01-01

    Roč. 276, Suppl.1 (2009), s. 346. ISSN 1742-464X. [FEBS Congress /34./. 04.07.2009-09.07.2009, Prague] Institutional research plan: CEZ:AV0Z40310501 Keywords : cardiac stem cells * secreted paracrine/autocrine factors * proteomics Subject RIV: CB - Analytical Chemistry, Separation

  9. Kinomic and phospho-proteomic analysis of breast cancer stem-like cells

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Christensen, Anne Geske Lindhard; Ehmsen, Sidse;

    Kinomic and phospho-proteomic analysis of breast cancer stem-like cells Rikke Leth-Larsen1, Anne G Christensen1, Sidse Ehmsen1, Mark Møller1, Giuseppe Palmisano2, Martin R Larsen2, Henrik J Ditzel1,3 1Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark 2Institute of...

  10. Identification and functionality of proteomes secreted by rat cardiac stem cells and neonatal cardiomyocytes

    Czech Academy of Sciences Publication Activity Database

    Šťastná, Miroslava; Chimenti, I.; Marban, E.; Van Eyk, J.E.

    2010-01-01

    Roč. 10, č. 2 (2010), s. 245-253. ISSN 1615-9853 Institutional research plan: CEZ:AV0Z40310501 Keywords : animal proteomics * cardiac stem cells * neonatal cardiomyocytes Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.815, year: 2010

  11. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T.G.; Prokhorova, Tatyana; Akimov, Vyacheslav;

    2011-01-01

    To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by...

  12. Proteomics analysis of human endothelial cells after shortterm exposure to mobile phone radiation

    International Nuclear Information System (INIS)

    Mobile phones have been a part of our everyday life in the developed world since the late 1990s. This has raised concerns over the potential health risks of mobile phone use. Biological and health effects potentially caused by mobile phone radiation have been extensively studied and several biological and medical endpoints have been examined. So far, results have not been conclusive on the potential effects of mobile phone radiation. Mobile phones generate a modulated radio frequency electromagnetic field (RF-EMF), which is a form of non-ionizing radiation. This means that mobile phone radiation does not have enough energy to ionize atoms and it cannot break chemical bonds directly (e.g., in DNA strands). There could, however, be other mechanisms by which mobile phone radiation may affect cellular and physiological functions. Whether these mechanisms exist is unknown. In this thesis, large-scale screening techniques, such as proteomics, were applied to examine changes on the proteome level after exposure to mobile phone radiation. Proteomics techniques allow the screening of several hundreds, and even thousands, of proteins simultaneously, and are thus more efficient than single endpoint techniques. Four different types of human endothelial cells (two cell lines, two types of primary cells) were exposed to two types of mobile phone radiation (900 and 1800 MHz GSM). The proteome of these cells was examined immediately after short-term exposure using two-dimensional gel electrophoresis (2DE). Two protein detection/analysis techniques were used: silver staining for the cell line samples and difference gel electrophoresis (DIGE) for the primary cells. 2DE-DIGE technology is currently a state-of-the-art technique in 2DE studies. Several changes were found in the proteome of the human endothelial cell line EA.hy926 after exposure to 900 MHz GSM mobile phone radiation. In addition, the proteome of a variant of the same cell line, the EA.hy926v1, was affected after 900 MHz

  13. Environmental proteomics – what proteins from soil and surface water can tell us: a perspective

    Directory of Open Access Journals (Sweden)

    W. Schulze

    2004-07-01

    Full Text Available Mass spectrometry based proteomics is widely used to study cellular processes in model organisms. However, it has not much been applied in environmental research because it was thought that free proteins would not be sufficiently stable in the environments. Based on recent observations that protein can readily be detected as a component of dissolve organic carbon, this article gives an overview about the possible use of proteomic methods in ecology and environmental sciences. At this stage, there are two areas of interest: (1 the identification of phylogenetic groups contributing to the DOC pool, and (2 identification of the origin of specific enzymes that are important for ecosystem processes. In this paper methods of mass spectrometry based proteomics were applied to identify proteins from DOC and water samples from different environments. It is demonstrated, that environmental proteomics is capable to distinguish the active set of organisms of different horizons of soils, and from various sources of surface water. Currently the limitation is given by the present knowledge of the genome of soil organisms. In addition, environmental proteomics allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific enzymes. Taking laccases as an example, it is shown that this enzyme is excreted into soils by a whole range of organisms from different phylogenetic groups. Further applications, such as in pollution reseach are conceivable. In summary, environmental proteomcis opens a new area of research between the fields of microbiology and biogeochemistry.

  14. Environmental proteomics what proteins from soil and surface water can tell us: a perspective

    Science.gov (United States)

    Schulze, W.

    2004-07-01

    Mass spectrometry based proteomics is widely used to study cellular processes in model organisms. However, it has not much been applied in environmental research because it was thought that free proteins would not be sufficiently stable in the environments. Based on recent observations that protein can readily be detected as a component of dissolve organic carbon, this article gives an overview about the possible use of proteomic methods in ecology and environmental sciences. At this stage, there are two areas of interest: (1) the identification of phylogenetic groups contributing to the DOC pool, and (2) identification of the origin of specific enzymes that are important for ecosystem processes. In this paper methods of mass spectrometry based proteomics were applied to identify proteins from DOC and water samples from different environments. It is demonstrated, that environmental proteomics is capable to distinguish the active set of organisms of different horizons of soils, and from various sources of surface water. Currently the limitation is given by the present knowledge of the genome of soil organisms. In addition, environmental proteomics allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific enzymes. Taking laccases as an example, it is shown that this enzyme is excreted into soils by a whole range of organisms from different phylogenetic groups. Further applications, such as in pollution reseach are conceivable. In summary, environmental proteomcis opens a new area of research between the fields of microbiology and biogeochemistry.

  15. Functional proteomics of barley and barley chloroplasts – strategies, methods and perspectives

    DEFF Research Database (Denmark)

    Petersen, Jørgen; Rogowska-Wrzesinska, Adelina; Jensen, Ole Nørregaard

    2013-01-01

    whole-genome sequencing of barley revealed approximately 26,100 open reading frames, which provides a foundation for detailed molecular studies of barley by functional genomics and proteomics approaches. Such studies will provide further insights into the mechanisms of, for example, drought and stress......Barley (Hordeum vulgare) is an important cereal grain that is used in a range of products for animal and human consumption. Crop yield and seed quality has been optimized during decades by plant breeding programs supported by biotechnology and molecular biology techniques. The recently completed...... tolerance, micronutrient utilization, and photosynthesis in barley. In the present review we present the current state of proteomics research for investigations of barley chloroplasts, i.e., the organelle that contain the photosynthetic apparatus in the plant. We describe several different proteomics...

  16. Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective

    Directory of Open Access Journals (Sweden)

    Olga Pechanova

    2015-11-01

    Full Text Available Maize (Zea mays L. is a host to numerous pathogenic species that impose serious diseases to its ear and foliage, negatively affecting the yield and the quality of the maize crop. A considerable amount of research has been carried out to elucidate mechanisms of maize-pathogen interactions with a major goal to identify defense-associated proteins. In this review, we summarize interactions of maize with its agriculturally important pathogens that were assessed at the proteome level. Employing differential analyses, such as the comparison of pathogen-resistant and susceptible maize varieties, as well as changes in maize proteomes after pathogen challenge, numerous proteins were identified as possible candidates in maize resistance. We describe findings of various research groups that used mainly mass spectrometry-based, high through-put proteomic tools to investigate maize interactions with fungal pathogens Aspergillus flavus, Fusarium spp., and Curvularia lunata, and viral agents Rice Black-streaked Dwarf Virus and Sugarcane Mosaic Virus.

  17. Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue

    Science.gov (United States)

    Mehrabian, Mohadeseh; Brethour, Dylan; Williams, Declan; Wang, Hansen; Arnould, Hélène; Schneider, Benoit; Schmitt-Ulms, Gerold

    2016-01-01

    A popular method for studying the function of a given protein is to generate and characterize a suitable model deficient for its expression. For the prion protein (PrP), best known for its role in several invariably fatal neurodegenerative diseases, a natural choice, therefore, would be to undertake such studies with brain samples. We recently documented the surprising observation that PrP deficiency caused a loss or enhancement of NCAM1 polysialylation, dependent on the cell model used. To identify possible causes for this disparity, we set out to systematically investigate the consequence of PrP deficiency on the global proteome in brain tissue and in four distinct cell models. Here we report that PrP deficiency causes robust but surprisingly divergent changes to the global proteomes of cell models but has no discernible impact on the global brain proteome. Amongst >1,500 proteins whose levels were compared in wild-type and PrP-deficient models, members of the MARCKS protein family exhibited pronounced, yet cell model-dependent changes to their steady-state levels. Follow-up experiments revealed that PrP collaborates with members of the MARCKS protein family in its control of NCAM1 polysialylation. We conclude that the physiological function of PrP may be masked in analyses of complex brain samples but its cell-type specific influence on a lipid raft-based NCAM1-related cell biology comes to the fore in investigations of specific cell types. PMID:27327609

  18. Contribution of MS-based proteomics to the understanding of Herpes Simplex Virus type 1 interaction with host cells

    Directory of Open Access Journals (Sweden)

    EnriqueSantamaría

    2012-03-01

    Full Text Available Like other DNA viruses, Herpes Simplex Virus type 1 (HSV-1 replicates and proliferates in host cells continuously modulating the host molecular environment. Following a sophisticated temporal expression pattern, HSV-1 encodes at least 89 multifunctional proteins that interplay with and modify the host cell proteome. During the last decade, advances in mass spectrometry applications coupled to the development of proteomic separation methods have allowed to partially monitor the impact of HSV-1 infection in human cells. In this review, we discuss the current use of different proteome fractionation strategies to define HSV-1 targets on two major application areas: i viral protein interactomics to decipher viral protein interactions in host cells and ii differential quantitative proteomics to analyse the virally induced changes in the cellular proteome. Moreover, we will also discuss the potential application of high throughput proteomic approaches to study global proteome dynamics and also post-translational modifications in HSV-1-infected cells, what will greatly improved our molecular knowledge of HSV-1 infection.

  19. Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kesavan Meganathan

    Full Text Available Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs. Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE coupled with Tandem Mass spectrometry to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s. Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3 after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2, that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

  20. Bovine neonatal pancytopenia - Comparative proteomic characterization of two BVD vaccines and the producer cell surface proteome (MDBK

    Directory of Open Access Journals (Sweden)

    Euler Kerstin N

    2013-01-01

    Full Text Available Abstract Background Bovine neonatal pancytopenia (BNP is a disease syndrome in newborn calves of up to four weeks of age, first observed in southern Germany in 2006. By now, cases have been reported in several countries around the globe. Many affected calves die within days due to multiple haemorrhages, thrombocytopenia, leukocytopenia and bone marrow depletion. A certain vaccine directed against Bovine Virus Diarrhoea Virus (BVDV was recently shown to be associated with BNP pathogenesis. Immunized cows develop alloantibodies that are transferred to newborn calves via colostrum intake. In order to further elucidate BNP pathogenesis, the purpose of this study was to characterize and compare the protein composition of the associated vaccine to another vaccine directed against BVDV not related to BNP and the cell surface proteome of MDBK (Madin-Darby Bovine Kidney cells, the cell line used for production of the associated vaccine. Results By SDS-PAGE and mass spectrometry, we were able to detect several coagulation-related and immune modulatory proteins, as well as cellular and serum derived molecules being shared between the associated vaccine and MDBK cells. Furthermore, the number of proteins identified in the BNP related vaccine was almost as high as the number of surface proteins detected on MDBK cells and exceeded the amount of proteins identified in the non-BNP related vaccine over 3.5 fold. The great amount of shared cellular and serum derived proteins confirm that the BNP associated vaccine contained many molecules originating from MDBK cells and vaccine production. Conclusions The respective vaccine was not purified enough to prevent the development of alloantibodies. To narrow down possible candidate proteins, those most likely to represent a trigger for BNP pathogenesis are presented in this study, giving a fundament for further analysis in future research.

  1. Analysis of membrane proteome and secretome in cells over-expressing ADAM17 using quantitative proteomics

    International Nuclear Information System (INIS)

    Full text: A disintegrin and metalloproteinase (ADAM) protease is involved in proteolytic ectodomain shedding of several membrane-associated proteins and modulation of key cell signaling pathways in the tumor microenvironment. In this study, we examined the effect of over-expressing the full length human ADAM17 in membrane and secreted proteins. To this end, we constructed a stable Flp-In T-RExHEK293 cells expressing ADAM17 by tetracycline induction. These cells were grown in Dulbeccos modified Eagles medium containing light lysine, arginine or heavy, L-Arg-13C615N4 and L-Lys -13C615N2 (SILAC: stable isotope labeling with amino acid in cell culture) media and they were treated with an ADAM17 activator, phorbolester (PMA). Controls such as Flp-In T-RExHEK293 cell without PMA treatment and without ADAM17 cloned were cultivated in light medium. The ADAM17 overexpression was induced with tetracycline 500 ng/ml for 24 hours. Cells in a heavy condition were treated with PMA 50 ng/ml for 1 hour and vehicle DMSO was used as control in a light cell condition. The extracellular media were collected, concentrated and used to evaluate the secretome and a cell surface biotinylation-based approach was used to capture cell surface-associated proteins. The biotinylated proteins were eluted with dithiothreitol, alkylated with iodoacetamide and then digested with trypsin. The resulting peptides were subjected to LC-MS/MS analysis on an ETD enabled Orbitrap Velos instrument. The results showed different proteins up or down regulated in membrane and secretome analysis which might represent potential molecules involved in signaling or ADAM17 regulation events. (author)

  2. Analysis of membrane proteome and secretome in cells over-expressing ADAM17 using quantitative proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, R.; Simabuco, F.M. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Yokoo, S.; Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Sherman, N. [University of Virginia, Charlottesville, VA (United States)

    2012-07-01

    Full text: A disintegrin and metalloproteinase (ADAM) protease is involved in proteolytic ectodomain shedding of several membrane-associated proteins and modulation of key cell signaling pathways in the tumor microenvironment. In this study, we examined the effect of over-expressing the full length human ADAM17 in membrane and secreted proteins. To this end, we constructed a stable Flp-In T-RExHEK293 cells expressing ADAM17 by tetracycline induction. These cells were grown in Dulbeccos modified Eagles medium containing light lysine, arginine or heavy, L-Arg-13C615N4 and L-Lys -13C615N2 (SILAC: stable isotope labeling with amino acid in cell culture) media and they were treated with an ADAM17 activator, phorbolester (PMA). Controls such as Flp-In T-RExHEK293 cell without PMA treatment and without ADAM17 cloned were cultivated in light medium. The ADAM17 overexpression was induced with tetracycline 500 ng/ml for 24 hours. Cells in a heavy condition were treated with PMA 50 ng/ml for 1 hour and vehicle DMSO was used as control in a light cell condition. The extracellular media were collected, concentrated and used to evaluate the secretome and a cell surface biotinylation-based approach was used to capture cell surface-associated proteins. The biotinylated proteins were eluted with dithiothreitol, alkylated with iodoacetamide and then digested with trypsin. The resulting peptides were subjected to LC-MS/MS analysis on an ETD enabled Orbitrap Velos instrument. The results showed different proteins up or down regulated in membrane and secretome analysis which might represent potential molecules involved in signaling or ADAM17 regulation events. (author)

  3. Proteomic Analysis of Terminalia chebula Extract-Dependent Changes in Human Lymphoblastic T Cell Protein Expression

    Science.gov (United States)

    Das, Nando Dulal; Jung, Kyoung Hwa; Park, Ji Hyun; Choi, Mi Ran; Lee, Hyung Tae; Kim, Moo Sung; Lee, Sang Rin

    2012-01-01

    Abstract Terminalia chebula is a native plant from southern Asia to southwestern China that is used in traditional medicine for the treatment of malignant tumors and diabetes. This plant also has antibacterial and immunomodulatory properties. The present study assessed T. chebula extract-dependent protein expression changes in Jurkat cells. Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry and Ingenuity Pathways Analysis (IPA) were performed to assess protein expression and networks, respectively. A comparative proteomic profile was determined in T. chebula extract (50 μg/mL)-treated and control cells; the expressions of β-tubulin, ring finger and CHY zinc finger domain containing 1, and insulin-like growth factor 1 receptor kinase were significantly down-regulated in T. chebula extract-treated Jurkat cells. Moreover, the molecular basis for the T. chebula extract-dependent protein expression changes in Jurkat cells was determined by IPA. Treatment with the T. chebula extract significantly inhibited nuclear factor-κB activity and affected the proteomic profile of Jurkat cells. The molecular network signatures and functional proteomics obtained in this study may facilitate the evaluation of potential antitumor therapeutic targets and elucidate the molecular mechanism of T. chebula extract-dependent effects in Jurkat cells. PMID:22471968

  4. In-Depth Profiling of the Peripheral Blood Mononuclear Cells Proteome for Clinical Blood Proteomics

    OpenAIRE

    Saša Končarević; Christopher Lößner; Karsten Kuhn; Thorsten Prinz; Ian Pike; Hans-Dieter Zucht

    2014-01-01

    Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and p...

  5. SILAC-Based Quantitative Proteomic Analysis of Human Lung Cell Response to Copper Oxide Nanoparticles

    OpenAIRE

    Edelmann, Mariola J.; Shack, Leslie A; Caitlin D Naske; Walters, Keisha B.; Nanduri, Bindu

    2014-01-01

    Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the...

  6. The Proteome of Native Adult Müller Glial Cells From Murine Retina.

    Science.gov (United States)

    Grosche, Antje; Hauser, Alexandra; Lepper, Marlen Franziska; Mayo, Rebecca; von Toerne, Christine; Merl-Pham, Juliane; Hauck, Stefanie M

    2016-02-01

    To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial

  7. Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.; Ari, Krakowski; Luo, Kunxin; Chen, David J.; Li, Song

    2004-08-08

    Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.

  8. Systems analysis of endothelial cell plasma membrane proteome of rat lung microvasculature

    Directory of Open Access Journals (Sweden)

    Witkiewicz Halina

    2011-03-01

    Full Text Available Abstract Background Endothelial cells line all blood vessels to form the blood-tissue interface which is critical for maintaining organ homeostasis and facilitates molecular exchange. We recently used tissue subcellular fractionation combined with several multi-dimensional mass spectrometry-based techniques to enhance identification of lipid-embedded proteins for large-scale proteomic mapping of luminal endothelial cell plasma membranes isolated directly from rat lungs in vivo. The biological processes and functions of the proteins expressed at this important blood-tissue interface remain unexplored at a large scale. Results We performed an unbiased systems analysis of the endothelial cell surface proteome containing over 1800 proteins to unravel the major functions and pathways apparent at this interface. As expected, many key functions of plasma membranes in general (i.e., cell surface signaling pathways, cytoskeletal organization, adhesion, membrane trafficking, metabolism, mechanotransduction, membrane fusion, and vesicle-mediated transport and endothelial cells in particular (i.e., blood vessel development and maturation, angiogenesis, regulation of endothelial cell proliferation, protease activity, and endocytosis were significantly overrepresented in this proteome. We found that endothelial cells express multiple proteins that mediate processes previously reported to be restricted to neuronal cells, such as neuronal survival and plasticity, axon growth and regeneration, synaptic vesicle trafficking and neurotransmitter metabolic process. Surprisingly, molecular machinery for protein synthesis was also detected as overrepresented, suggesting that endothelial cells, like neurons, can synthesize proteins locally at the cell surface. Conclusion Our unbiased systems analysis has led to the potential discovery of unexpected functions in normal endothelium. The discovery of the existence of protein synthesis at the plasma membrane in endothelial

  9. Plant cell wall proteomics: the leadership of Arabidopsis thaliana

    OpenAIRE

    Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

    2013-01-01

    Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell wa...

  10. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

    OpenAIRE

    Xin Deng, Jian Liang, Zhi-Xiu Lin, Fa-Sheng Wu, Ya-Ping Zhang, Zhi-Wei Zhang

    2010-01-01

    AIM: To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.METHODS: A proteomic strategy combining two-dimensional gel electrophoresis and ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) was used to study the differential expression of proteins and Western blotting was use...

  11. Proteomic profiling of a robust Wolbachia infection in an Aedes albopictus mosquito cell line

    OpenAIRE

    Baldridge, Gerald D; Baldridge, Abigail S.; Witthuhn, Bruce A.; Higgins, LeeAnn; Markowski, Todd W.; FALLON, ANN M.

    2014-01-01

    Wolbachia pipientis a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein “footprint” ...

  12. Traumatically injured astrocytes release a proteomic signature modulated by STAT3-dependent cell survival.

    Science.gov (United States)

    Levine, Jaclynn; Kwon, Eunice; Paez, Pablo; Yan, Weihong; Czerwieniec, Gregg; Loo, Joseph A; Sofroniew, Michael V; Wanner, Ina-Beate

    2016-05-01

    Molecular markers associated with CNS injury are of diagnostic interest. Mechanical trauma generates cellular deformation associated with membrane permeability with unknown molecular consequences. We used an in vitro model of stretch-injury and proteomic analyses to determine protein changes in murine astrocytes and their surrounding fluids. Abrupt pressure-pulse stretching resulted in the rapid release of 59 astrocytic proteins with profiles reflecting cell injury and cell death, i.e., mechanoporation and cell lysis. This acute trauma-release proteome was overrepresented with metabolic proteins compared with the uninjured cellular proteome, bearing relevance for post-traumatic metabolic depression. Astrocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3-CKO) resulted in reduced stretch-injury tolerance, elevated necrosis and increased protein release. Consistent with more lysed cells, more protein complexes, nuclear and transport proteins were released from STAT3-CKO versus nontransgenic astrocytes. STAT3-CKO astrocytes had reduced basal expression of GFAP, lactate dehydrogenase B (LDHB), aldolase C (ALDOC), and astrocytic phosphoprotein 15 (PEA15), and elevated levels of tropomyosin (TPM4) and α actinin 4 (ACTN4). Stretching caused STAT3-dependent cellular depletion of PEA15 and GFAP, and its filament disassembly in subpopulations of injured astrocytes. PEA15 and ALDOC signals were low in injured astrocytes acutely after mouse spinal cord crush injury and were robustly expressed in reactive astrocytes 1 day postinjury. In contrast, α crystallin (CRYAB) was present in acutely injured astrocytes, and absent from uninjured and reactive astrocytes, demonstrating novel marker differences among postinjury astrocytes. These findings reveal a proteomic signature of traumatically-injured astrocytes reflecting STAT3-dependent cellular survival with potential diagnostic value. GLIA 2016;64:668-694. PMID:26683444

  13. Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

    OpenAIRE

    Kesavan Meganathan; Smita Jagtap; Vilas Wagh; Johannes Winkler; John Antonydas Gaspar; Diana Hildebrand; Maria Trusch; Karola Lehmann; Jürgen Hescheler; Hartmut Schlüter; Agapios Sachinidis

    2012-01-01

    Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics prof...

  14. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.METHODS: A proteomic strategy combining two-dimensional gel electrophoresis and ultraperform ance liquid chromatographyelectrospray ionizationtandem mass spectrometry (UPLCESIMS/MS) was used to study the differential expression of proteins and Western blotting was used to validate the re...

  15. Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Madsen, Søren M; Glenting, Jacob;

    2009-01-01

    In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel...... probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics....

  16. Proteomics in China:Ready for prime time

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Proteomics is a newborn science focusing on the comprehensive systematic analysis of all proteins in molecule machineries,organelles,cells,tissues,organs or intact organisms.It has been becoming one of the focuses in life sciences and cutting-edge techniques in biotechnologies in the 21st century.During the last decade,proteomics in China has developed much faster than other developing fields in the life sciences.This review article briefly retrospects the origin and development of proteomics in China,and provides an overview of representative scientific progress and perspectives.

  17. Application for proteomic techniques in studying osteoarthritis: a review

    Directory of Open Access Journals (Sweden)

    YvesHenrotin

    2011-12-01

    Full Text Available After the genomic era, proteomic corresponds to a wide variety of techniques to study the protein content of cells, tissue or organism and to isolate protein of interest. It offers the choice between gel-based and gel-free methods or shotgun proteomics. Applications of proteomic technology may concern three principal objectives in several biomedical or clinical domains of research as in osteoarthritis: (i to understand the physiopathology and underlying mechanisms leading to a disease or associated to a particular model, (ii, to find disease-specific biomarker and (iii to identify new therapeutic targets. This review aimed at gathering most of the data regarding the proteomic techniques and their applications to arthritis research. It also reported technical limitations and solutions, as for example for sample preparation. Proteomics open wide perspectives in biochemical research but many technical matters still remain to be solved.

  18. Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

    Directory of Open Access Journals (Sweden)

    Ruili Ma

    Full Text Available The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV-infected swine testicular (ST cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1, caspase-8, and heat shock protein 90 alpha (HSP90α were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

  19. Synergistic effects of retinoic acid and tamoxifen on human breast cancer cells: Proteomic characterization

    International Nuclear Information System (INIS)

    The anti-estrogen tamoxifen and vitamin A-related compound, all-trans retinoic acid (RA), in combination act synergistically to inhibit the growth of MCF-7 human breast cancer cells. In the present study, we applied two-dimensional gel electrophoresis based proteomic approach to globally analyze this synergistic effect of RA and tamoxifen. Proteomic study revealed that multiple clusters of proteins were involved in RA and tamoxifen-induced apoptosis in MCF-7 breast cancer cells, including post-transcriptional and splicing factors, proteins related to cellular proliferation or differentiation, and proteins related to energy production and internal degradation systems. The negative growth factor-transforming growth factor β (TGFβ) was secreted by RA and/or tamoxifen treatment and was studies as a potential mediator of the synergistic effects of RA and tamoxifen in apoptosis. By comparing protein alterations in treatments of RA and tamoxifen alone or in combination to those of TGFβ treatment, or co-treatment with TGFβ inhibitor SB 431542, proteomic results showed that a number of proteins were involved in TGFβ signaling pathway. These results provide valuable insights into the mechanisms of RA and tamoxifen-induced TGFβ signaling pathway in breast cancer cells

  20. Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method.

    Directory of Open Access Journals (Sweden)

    Ganglong Yang

    Full Text Available The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia, KK47 (low grade nonmuscle invasive bladder cancer, NMIBC, and YTS1 (metastatic bladder cancer have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.

  1. Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

    Directory of Open Access Journals (Sweden)

    Yoichiro Fukao

    2016-01-01

    Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

  2. Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

    International Nuclear Information System (INIS)

    Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs

  3. Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

    Energy Technology Data Exchange (ETDEWEB)

    Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Lee, Yeon-Mi [Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Park, Bong-Wook; Byun, June-Ho [Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju 660-702 (Korea, Republic of); Ahn, Chun-Seob; Kim, Jae-Won [Department of Microbiology, Division of Life Sciences, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Rho, Gyu-Jin, E-mail: jinrho@gnu.ac.kr [Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701 (Korea, Republic of)

    2014-01-01

    Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.

  4. Proteomic analysis of human blastocoel fluid and blastocyst cells

    DEFF Research Database (Denmark)

    Jensen, Pernille; Beck, Hans Christian; Petersen, Jørgen;

    2013-01-01

    Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst and can differentiate into any cell type in the human body. These cells hold a great potential for regenerative medicine, but to obtain enough cells needed for medical treatment, culture is required on a...... blastocoel fluid, which is in contact with all the cells in the blastocyst, including hESCs. Fifty-three surplus human blastocysts were donated after informed consent, and blastocoel fluid was isolated by micromanipulation. Using highly sensitive nano-high-pressure liquid chromatography-tandem mass...

  5. Proteomics Core

    Data.gov (United States)

    Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in...

  6. Plant cell wall proteomics: mass spectrometry data, a trove for research on protein structure/function relationships.

    OpenAIRE

    Albenne, Cécile; Canut, Hervé; Boudart, Georges; Zhang, Yu; San Clemente, Hélène; Pont-Lezica, Rafael; Jamet, Elisabeth

    2009-01-01

    International audience Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organelles. In particular, mass spectrometry (MS) analysis of gel-separated proteins produces data not only for protein identification, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics of Arabidopsis thaliana. These analyses show that highly homologous members of ...

  7. Modulation of the proteome of peripheral blood mononuclear cells from HIV-1 infected patients by drugs of abuse

    OpenAIRE

    Jessica L. Reynolds; Supriya D Mahajan; Aalinkeel, Ravikunar; Nair, Bindukumar; Sykes, Donald E; Agosto-Mujica, Arnadri; Hsiao, Chiu Bin; Schwartz, Stanley A.

    2009-01-01

    We used proteomic analyses to assess how drug abuse modulates immunologic responses to infections with the human immunodeficiency virus type 1 (HIV-1). Two dimensional (2D) difference gel electrophoresis was utilized to determine changes in the proteome of peripheral blood mononuclear cells (PBMC) isolated from HIV-1 positive donors that occurred after treatment with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins. We fu...

  8. Human lung epithelial cell A549 proteome data after treatment with titanium dioxide and carbon black.

    Science.gov (United States)

    Vuong, Ngoc Q; Goegan, Patrick; Mohottalage, Susantha; Breznan, Dalibor; Ariganello, Marianne; Williams, Andrew; Elisma, Fred; Karthikeyan, Subramanian; Vincent, Renaud; Kumarathasan, Premkumari

    2016-09-01

    Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, "Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures" (Vuong et al., 2016) [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures. PMID:27508218

  9. Label-free quantitative proteomics of CD133-positive liver cancer stem cells

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    Tsai Sheng-Ta

    2012-11-01

    Full Text Available Abstract Background CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q and MaxQuant. Results Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. Conclusions These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

  10. Comparative membrane proteomics: a technical advancement in the search of renal cell carcinoma biomarkers.

    Science.gov (United States)

    Raimondo, Francesca; Corbetta, Samuele; Savoia, Andrea; Chinello, Clizia; Cazzaniga, Marta; Rocco, Francesco; Bosari, Silvano; Grasso, Marco; Bovo, Giorgio; Magni, Fulvio; Pitto, Marina

    2015-06-01

    Renal Cell Carcinoma (RCC) is the most common kidney cancer, accounting for 3% of adult malignancies, with high metastatic potential and radio-/chemo-resistance. To investigate the protein profile of membrane microdomains (MD), plasma membrane supramolecular structures involved in cell signaling, transport, and neoplastic transformation, we set up a proteomic bottom-up approach as a starting point for the identification of potential RCC biomarkers. We purified MD from RCC and adjacent normal kidney (ANK) tissues, through their resistance to non-ionic detergents followed by ultracentrifugation in sucrose density gradient. MD from 5 RCC/ANK tissues were then pooled and analysed by LC-ESI-MS/MS. In order to identify the highest number of proteins and increase the amount of membrane and hydrophobic ones, we first optimized an enzymatic digestion protocol based on Filter Aided Sample Preparation (FASP), coupled to MD delipidation. The MS analysis led to the identification of 742 ANK MD and 721 RCC MD proteins, of which, respectively, 53.1% and 52.6% were membrane- bound. Additionally, we evaluated RCC MD differential proteome by label-free quantification; 170 and 126 proteins were found to be, respectively, up-regulated and down-regulated in RCC MD. Some differential proteins, namely CA2, CD13, and ANXA2, were subjected to validation by immunodecoration. These results show the importance of setting up different protocols for the proteomic analysis of membrane proteins, specific to the different molecular features of the samples. Furthermore, the subcellular proteomic approach provided a list of differentially expressed proteins among which RCC biomarkers may be looked for. PMID:25926002

  11. Changes in the proteomic profile of adipose tissue-derived mesenchymal stem cells during passages

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    Capra Emanuele

    2012-07-01

    Full Text Available Abstract Background Human mesenchymal stem cells (hMSC have recently raised the attention because of their therapeutic potential in the novel context of regenerative medicine. However, the safety of these new and promising cellular products should be carefully defined before they can be used in the clinical setting, as. The protein expression profile of these cells might reveal potential hazards associated with senescence and tumoral transformation which may occur during culture. Proteomic is a valuable tool for hMSC characterization and identification of possible changes during expansion. Results We used Surface Enhanced Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry (SELDI-ToF-MS to evaluate the presence of stable molecular markers in adipose tissue-derived mesenchymal stem cells (AD-MSC produced under conditions of good manufacturing practices (GMP. Proteomic patterns of cells prepared were consistent, with 4 up-regulated peaks (mass-to-charge ratio (m/z 8950, 10087, 10345, and 13058 through subculture steps (P0-P7 with similar trend in three donors. Among the differentially expressed proteins found in the cytoplasmic and nuclear fractions, a cytoplasmic 10.1 kDa protein was upregulated during culture passages and was identified as S100A6 (Calcyclin. Conclusions This study suggests for the first time that common variation could occur in AD-MSC from different donors, with the identification of S100A6, a protein prevalently related to cell proliferation and cell culture condition. These results support the hypothesis of common proteomic changes during MSCs expansion and could give important insight in the knowledge of molecular mechanisms intervening during MSC expansion.

  12. New perspectives on host-parasite interplay by comparative transcriptomic and proteomic analyses of Schistosoma japonicum.

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    Feng Liu

    2006-04-01

    Full Text Available Schistosomiasis remains a serious public health problem with an estimated 200 million people infected in 76 countries. Here we isolated ~ 8,400 potential protein-encoding cDNA contigs from Schistosoma japonicum after sequencing circa 84,000 expressed sequence tags. In tandem, we undertook a high-throughput proteomics approach to characterize the protein expression profiles of a number of developmental stages (cercariae, hepatic schistosomula, female and male adults, eggs, and miracidia and tissues at the host-parasite interface (eggshell and tegument by interrogating the protein database deduced from the contigs. Comparative analysis of these transcriptomic and proteomic data, the latter including 3,260 proteins with putative identities, revealed differential expression of genes among the various developmental stages and sexes of S. japonicum and localization of putative secretory and membrane antigens, enzymes, and other gene products on the adult tegument and eggshell, many of which displayed genetic polymorphisms. Numerous S. japonicum genes exhibited high levels of identity with those of their mammalian hosts, whereas many others appeared to be conserved only across the genus Schistosoma or Phylum Platyhelminthes. These findings are expected to provide new insights into the pathophysiology of schistosomiasis and for the development of improved interventions for disease control and will facilitate a more fundamental understanding of schistosome biology, evolution, and the host-parasite interplay.

  13. T-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome

    DEFF Research Database (Denmark)

    Bresciani, Anne Gøther; Paul, Sinu; Schommer, Nina;

    2016-01-01

    allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T-cell epitopes, we found that epitopes that are similar with self-antigens above a certain threshold showed lower immunogenicity, presumably...... as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and...

  14. Biochemistry, proteomics and phosphoproteomics of plant mitochondria from non-photosynthetic cells

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    Jesper Foged Havelund

    2013-03-01

    Full Text Available Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(PH and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified.

  15. Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells.

    Science.gov (United States)

    Ahmed, Nuzhat; Greening, David; Samardzija, Chantel; Escalona, Ruth M; Chen, Maoshan; Findlay, Jock K; Kannourakis, George

    2016-01-01

    Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission after initial surgery and chemotherapy. However, most patients die within identification of 353 proteins. There were significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells. In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cells from CN and CR ovarian cancer patients. PMID:27470985

  16. A draft map of the mouse pluripotent stem cell spatial proteome.

    Science.gov (United States)

    Christoforou, Andy; Mulvey, Claire M; Breckels, Lisa M; Geladaki, Aikaterini; Hurrell, Tracey; Hayward, Penelope C; Naake, Thomas; Gatto, Laurent; Viner, Rosa; Martinez Arias, Alfonso; Lilley, Kathryn S

    2016-01-01

    Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data. PMID:26754106

  17. Proteomic changes resulting from gene copy number variations in cancer cells.

    Directory of Open Access Journals (Sweden)

    Tamar Geiger

    2010-09-01

    Full Text Available Along the transformation process, cells accumulate DNA aberrations, including mutations, translocations, amplifications, and deletions. Despite numerous studies, the overall effects of amplifications and deletions on the end point of gene expression--the level of proteins--is generally unknown. Here we use large-scale and high-resolution proteomics combined with gene copy number analysis to investigate in a global manner to what extent these genomic changes have a proteomic output and therefore the ability to affect cellular transformation. We accurately measure expression levels of 6,735 proteins and directly compare them to the gene copy number. We find that the average effect of these alterations on the protein expression is only a few percent. Nevertheless, by using a novel algorithm, we find the combined impact that many of these regional chromosomal aberrations have at the protein level. We show that proteins encoded by amplified oncogenes are often overexpressed, while adjacent amplified genes, which presumably do not promote growth and survival, are attenuated. Furthermore, regulation of biological processes and molecular complexes is independent of general copy number changes. By connecting the primary genome alteration to their proteomic consequences, this approach helps to interpret the data from large-scale cancer genomics efforts.

  18. SILAC-Based Quantitative Proteomic Analysis of Diffuse Large B-Cell Lymphoma Patients

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    Ulla Rüetschi

    2015-01-01

    Full Text Available Diffuse large B-cell lymphoma (DLBCL, the most common lymphoma, is a heterogeneous disease where the outcome for patients with early relapse or refractory disease is very poor, even in the era of immunochemotherapy. In order to describe possible differences in global protein expression and network patterns, we performed a SILAC-based shotgun (LC-MS/MS quantitative proteomic analysis in fresh-frozen tumor tissue from two groups of DLBCL patients with totally different clinical outcome: (i early relapsed or refractory and (ii long-term progression-free patients. We could identify over 3,500 proteins; more than 1,300 were quantified in all patients and 87 were significantly differentially expressed. By functional annotation analysis on the 66 proteins overexpressed in the progression-free patient group, we found an enrichment of proteins involved in the regulation and organization of the actin cytoskeleton. Also, five proteins from actin cytoskeleton regulation, applied in a supervised regression analysis, could discriminate the two patient groups. In conclusion, SILAC-based shotgun quantitative proteomic analysis appears to be a powerful tool to explore the proteome in DLBCL tumor tissue. Also, as progression-free patients had a higher expression of proteins involved in the actin cytoskeleton protein network, such a pattern indicates a functional role in the sustained response to immunochemotherapy.

  19. The Genomic and Proteomic Content of Cancer Cell-Derived Exosomes

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    Meredith C Henderson

    2012-04-01

    Full Text Available Exosomes are secreted membrane vesicles that have been proposed as an effective means to detect a variety of disease states, including cancer. The properties of exosomes, including stability in biological fluids, allow for their efficient isolation and make them an ideal vehicle for studies on early disease detection and evaluation. Much data has been collected over recent years regarding the mRNA, miRNA, and protein contents of exosomes. In addition, many studies have described the functional role that exosomes play in disease initiation and progression. Tumor cells have been shown to secrete exosomes, often in increased amounts compared to normal cells, and these exosomes can carry the genomic and proteomic signatures characteristic of the tumor cells from which they were derived. While these unique signatures make exosomes ideal for cancer detection, exosomes derived from cancer cells have also been shown to play a functional role in cancer progression. Here, we review the unique genomic and proteomic contents of exosomes originating from cancer cells as well as their functional effects to promote tumor progression.

  20. Proteomics study of progeny of normal human liver cells irradiated by 60Co γ-rays

    International Nuclear Information System (INIS)

    Objective: To characterize the differential protein expression in the progeny of human liver cells surviving from ionizing radiation by the proteomic analysis. Methods: Two-dimensional electrophoresis gel coupled with mass spectrometry was used to explore the specific protein expression in the progeny of 7702 human liver cells surviving from ionizing radiation. Alterations in expression level of protein spots between the control and the progeny groups were statistically analyzed by ImageMaster 2D Platinum software and mass spectrometry was used to identify the protein spots with significantly altered expression-level. Results: The progeny of irradiated ceils were derived from human liver cell line exposed to 0, 2, 4, 6 Gy of 60Co γ-irradiation. A total of 42 differentially expressed proteins between the control and the progeny of the irradiated cells groups were screened, of which 17 were identified by matrix assistant laser desorption ion-top off light-mass spectrometry (MALDI-TOF MS) analysis, including 4 up-regulated and 13 down-regnlated proteins. Conclusions: The differentially expressed proteins profile could be significantly altered in the progeny of irradiated cells. The proteomics approach has the potential to detect the protein changes relevant to radiatian-induced genomic instability (RIGI). Further study of differentially expressed proteins would likely reveal the molecular mechanisms of gene expression in RIGI. (authors)

  1. Transchromosomic cell model of Down syndrome shows aberrant migration, adhesion and proteome response to extracellular matrix

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    Cotter Finbarr E

    2009-08-01

    Full Text Available Abstract Background Down syndrome (DS, caused by trisomy of human chromosome 21 (HSA21, is the most common genetic birth defect. Congenital heart defects (CHD are seen in 40% of DS children, and >50% of all atrioventricular canal defects in infancy are caused by trisomy 21, but the causative genes remain unknown. Results Here we show that aberrant adhesion and proliferation of DS cells can be reproduced using a transchromosomic model of DS (mouse fibroblasts bearing supernumerary HSA21. We also demonstrate a deacrease of cell migration in transchromosomic cells independently of their adhesion properties. We show that cell-autonomous proteome response to the presence of Collagen VI in extracellular matrix is strongly affected by trisomy 21. Conclusion This set of experiments establishes a new model system for genetic dissection of the specific HSA21 gene-overdose contributions to aberrant cell migration, adhesion, proliferation and specific proteome response to collagen VI, cellular phenotypes linked to the pathogenesis of CHD.

  2. Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Kristin Surmann

    2016-06-01

    Full Text Available To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP encoding a continuously expressed green fluorescent protein (GFP. Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed. Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC–MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]. They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.

  3. Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

    Directory of Open Access Journals (Sweden)

    Canut Hervé

    2006-05-01

    Full Text Available Abstract Background The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP during the isolation procedure, (ii polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50% of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins. Results The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i homogenization in low ionic strength acid buffer to retain CWP, (ii purification through increasing density cushions, (iii extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%, belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification. Conclusion The

  4. Functional proteomics screen enables enrichment of distinct cell types from human pancreatic islets.

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    Revital Sharivkin

    Full Text Available The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9high/CD56+, glucagon-producing alpha cells (CD9-/CD56+ and trypsin-producing acinar cells (CD9-/CD56-. This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples.

  5. SILAC-based quantitative proteomic analysis of human lung cell response to copper oxide nanoparticles.

    Directory of Open Access Journals (Sweden)

    Mariola J Edelmann

    Full Text Available Copper (II oxide (CuO nanoparticles (NP are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

  6. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Dun [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Chen, Hai-Xiao, E-mail: Hxchen-1@163.net [Orthopedic Department, Taizhou Hospital, Wenzhou Medical College, Linhai, Zhejiang 317000 (China); Yu, Hai-Qiang [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Liang, Yong; Wang, Carrie [Population Council, 1230 York Avenue, New York, NY 10065 (United States); Lian, Qing-Quan [The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China); Deng, Hai-Teng, E-mail: dengh@mail.rockefeller.edu [Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ge, Ren-Shan, E-mail: rge@popcbr.rockefeller.edu [Population Council, 1230 York Avenue, New York, NY 10065 (United States); The 2nd Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang 325000 (China)

    2010-08-15

    Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

  7. Proteome analysis of virus-host cell interaction: rabies virus replication in Vero cells in two different media.

    Science.gov (United States)

    Kluge, Sabine; Rourou, Samia; Vester, Diana; Majoul, Samy; Benndorf, Dirk; Genzel, Yvonne; Rapp, Erdmann; Kallel, Héla; Reichl, Udo

    2013-06-01

    The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection. PMID:23674149

  8. Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomic studies.

    Science.gov (United States)

    Zhang, Ying; Bottinelli, Dario; Lisacek, Frédérique; Luban, Jeremy; Strambio-De-Castillia, Caterina; Varesio, Emmanuel; Hopfgartner, Gérard

    2015-09-01

    Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry (MS)-based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize MS coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilization and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA (radioimmunoprecipitation assay) buffer, was shown to be the method of choice based on total protein extraction and on the solubilization and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than that by 10% trichloroacetic acid (TCA)/acetone, allowing in excess of 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate into the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6 to 11% more distinct peptides and 14 to 19% more total proteins identified than using 0.5M triethylammonium bicarbonate alone, with the greatest increase (34%) for hydrophobic proteins. PMID:25983236

  9. Optimization of human dendritic cell sample preparation for mass spectrometry-based proteomics studies

    Science.gov (United States)

    Zhang, Ying; Bottinelli, Dario; Lisacek, Frédérique; Luban, Jeremy; De Castillia, Caterina Strambio; Varesio, Emmanuel; Hopfgartner, Gérard

    2016-01-01

    Dendritic cells (DCs) are specialized leukocytes that orchestrate the adaptive immune response. Mass spectrometry based proteomic study of these cells presents technical challenges, especially when the DCs are human in origin due to the paucity of available biological material. Here, to maximize mass spectrometry coverage of the global human DC proteome, different cell disruption methods, lysis conditions, protein precipitation, and protein pellet solubilisation and denaturation methods were compared. Mechanical disruption of DC cell pellets under cryogenic conditions, coupled with the use of RIPA buffer, was shown to be the method of choice based on total protein extraction and on the solubilisation and identification of nuclear proteins. Precipitation by acetone was found to be more efficient than by 10% TCA/acetone, allowing greater than 28% more protein identifications. Although being an effective strategy to eliminate the detergent residue, the acetone-wash step caused a loss of protein identifications. However, this potential drawback was overcome by adding 1% sodium deoxycholate in the dissolution buffer, which enhanced both solubility of the precipitated proteins and digestion efficiency. This in turn resulted in 6-11% more distinct peptides and 14-19% more total proteins identified than using 0.5M triethylammonium bicarbonate alone with the greatest increase (34%) for hydrophobic proteins. PMID:25983236

  10. Proteomic analysis of cervical cancer cells treated with suberonylanilide hydroxamic acid

    Indian Academy of Sciences (India)

    Jianxiong He; Canhua Huang; Aiping Tong; Bin Chen; Zhi Zeng; Peng Zhang; Chunting Wang; Yuquan Wei

    2008-12-01

    Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumour activity in a variety of tumour cells. To identify proteins involved in its antitumour activity, we utilized a proteomic approach to reveal protein expression changes in the human cervical cancer cell line HeLa following SAHA treatment. Protein expression profiles were analysed by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and protein identification was performed on a MALDI-Q-TOF MS/MS instrument. As a result, a total of nine differentially expressed proteins were visualized by 2-DE and Coomassie brilliant blue (CBB) staining. Further, all the changed proteins were positively identified via mass spectrometry (MS)/MS analysis. Of these, PGAM1 was significantly downregulated in HeLa cells after treatment with SAHA. Moreover, PGAM1 has been proven to be downregulated in another cervical cancer cell line (CaSki) by western blot analysis. Together, using proteomic tools, we identified several differentially expressed proteins that underwent SAHA-induced apoptosis. These changed proteins may provide some clues to a better understanding of the molecular mechanisms underlying SAHA-induced apoptosis in cervical cancer.

  11. Post-translational modifications of plant cell wall proteins and peptides: A survey from a proteomics point of view.

    Science.gov (United States)

    Canut, Hervé; Albenne, Cécile; Jamet, Elisabeth

    2016-08-01

    Plant cell wall proteins (CWPs) and peptides are important players in cell walls contributing to their assembly and their remodeling during development and in response to environmental constraints. Since the rise of proteomics technologies at the beginning of the 2000's, the knowledge of CWPs has greatly increased leading to the discovery of new CWP families and to the description of the cell wall proteomes of different organs of many plants. Conversely, cell wall peptidomics data are still lacking. In addition to the identification of CWPs and peptides by mass spectrometry (MS) and bioinformatics, proteomics has allowed to describe their post-translational modifications (PTMs). At present, the best known PTMs consist in proteolytic cleavage, N-glycosylation, hydroxylation of P residues into hydroxyproline residues (O), O-glycosylation and glypiation. In this review, the methods allowing the capture of the modified proteins based on the specific properties of their PTMs as well as the MS technologies used for their characterization are briefly described. A focus is done on proteolytic cleavage leading to protein maturation or release of signaling peptides and on O-glycosylation. Some new technologies, like top-down proteomics and terminomics, are described. They aim at a finer description of proteoforms resulting from PTMs or degradation mechanisms. This article is part of a Special Issue entitled: Plant Proteomics- a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. PMID:26945515

  12. Proteomic analysis of exosomes secreted by human mesothelioma cells

    NARCIS (Netherlands)

    J.P.J.J. Hegmans (Joost); A. Hemmes (Annabrita); T.M. Luider (Theo); M.J. Kleijmeer (Monique); J-B. Prins (Jan-Bas); L. Zitvogel; S.A. Burgers (Sjaak); H.C. Hoogsteden (Henk); B.N.M. Lambrecht (Bart); M.P.L. Bard (Martin)

    2004-01-01

    textabstractExosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins pre

  13. Neural stem cells and proteomics assesment of their properties

    Czech Academy of Sciences Publication Activity Database

    Skalníková, Helena; Halada, Petr; Vodička, Petr; Motlík, Jan; Kovářová, Hana

    Vídeň : FENS (The Federation of European Neuroscience Societies), 2006. s. 1. [Forum of European Neuroscience /5./. 08.07.2006-12.07.2006, Vienna] R&D Projects: GA MŠk LN00A065 Institutional research plan: CEZ:AV0Z50450515 Keywords : neural stem cells Subject RIV: EB - Genetics ; Molecular Biology

  14. Proteomics of membrane microdomains from activated human natural killer cells

    Czech Academy of Sciences Publication Activity Database

    Man, Petr; Pompach, Petr; Vančurová, Markéta; Novák, Petr; Kavan, Daniel; Mrázek, Hynek; Bezouška, Karel

    Bremen : German Society for Mass Spectrometry, 2009. s. 128-128. [International Mass Spectrometry Conference /18./. 30.08.2009-04.09.2009, Bremen] R&D Projects: GA MŠk LC07017; GA AV ČR KJB500200612 Institutional research plan: CEZ:AV0Z50200510 Keywords : mass spectrometry * natural killer cells Subject RIV: EE - Microbiology, Virology

  15. A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

    Directory of Open Access Journals (Sweden)

    Meike Herget

    Full Text Available Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

  16. Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells

    International Nuclear Information System (INIS)

    Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 μM benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10 μM) using this proteomics-based 2-D gel electrophoresis assay. We found 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them, 79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1 μM and 10 μM BaP-treated groups, 2 in the 0.1 μM and 10 μM BaP-treated groups, 4 in the 0.1 μM and 1 μM BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography–tandem mass spectrometry (LC–MS/MS) identification, and of these, 39 were identified, including subunits of the 26S proteasome and Annexin A1. The functions of some identified proteins were further examined and the results showed that they might be involved in BaP-induced DDR. Taken together, these data indicate that proteomics is a valuable approach in the study of environmental chemical–host interactions, and the identified proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis.

  17. HUMAN PLASMA PROTEOME MAPPING IN HEALTH AND CLEAR CELL CARCINOMA OF THE KIDNEY

    Directory of Open Access Journals (Sweden)

    V. E. Shevchenko

    2014-08-01

    Full Text Available Plasma proteome from patients with renal cell carcinoma (RCC and controls underwent mass spectrometric mapping. A total of 247 proteins were identified; the expression of 12 proteins of them increased on transition from the controls to patients with Stages I–II and III–IV RCC. There was decreased expression of 14 proteins in this series. Out of the 26 proteins showing a change in their expressions, 7 proteins belong to acute-phase ones, 3 proteins are associated with the intercellular matrix. These proteins can be potential markers for RCC.

  18. Proteomic analysis of mammalian sperm cells identifies new components of the centrosome

    OpenAIRE

    Fırat, Elif Nur Karalar; Sante, Joshua; Elliott, Sarah; Stearns, Tim

    2014-01-01

    1 Proteomic analysis of mammalian sperm cells identifies new components of the centrosome Elif N. Firat-Karalar1,3, Joshua Sante1,4, Sarah Elliott1, and Tim Stearns1,2 1Department of Biology, Stanford University, Stanford, CA 94305-5020, USA 2Department of Genetics, Stanford School of Medicine, Stanford, CA 94305-5120, USA 3Current address: Department of Molecular Biology and Genetics, Koç University, Istanbul, 34450, Turkey 4Current address: Division of Pulmonary and ...

  19. Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

    Science.gov (United States)

    Jayaramaiah, Usharani; Singh, Neetu; Thankappan, Sabarinath; Mohanty, Ashok Kumar; Chaudhuri, Pallab; Singh, Vijendra Pal; Nagaleekar, Viswas Konasagara

    2016-06-01

    Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates. PMID:26971466

  20. SILAC Proteomics of Planarians Identifies Ncoa5 as a Conserved Component of Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Alexander Böser

    2013-11-01

    Full Text Available Planarian regeneration depends on the presence of pluripotent stem cells in the adult. We developed an in vivo stable isotope labeling by amino acids in cell culture (SILAC protocol in planarians to identify proteins that are enriched in planarian stem cells. Through a comparison of SILAC proteomes of normal and stem cell-depleted planarians and of a stem cell-enriched population of sorted cells, we identified hundreds of stem cell proteins. One of these is an ortholog of nuclear receptor coactivator-5 (Ncoa5/CIA, which is known to regulate estrogen-receptor-mediated transcription in human cells. We show that Ncoa5 is essential for the maintenance of the pluripotent stem cell population in planarians and that a putative mouse ortholog is expressed in pluripotent cells of the embryo. Our study thus identifies a conserved component of pluripotent stem cells, demonstrating that planarians, in particular, when combined with in vivo SILAC, are a powerful model in stem cell research.

  1. Proteomic profiling of a robust Wolbachia infection in an Aedes albopictus mosquito cell line.

    Science.gov (United States)

    Baldridge, Gerald D; Baldridge, Abigail S; Witthuhn, Bruce A; Higgins, LeeAnn; Markowski, Todd W; Fallon, Ann M

    2014-11-01

    Wolbachia pipientis, a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein 'footprint' dominated by Wolbachia-encoded chaperones, stress response and cell membrane proteins, including the surface antigen WspA, a peptidoglycan-associated lipoprotein and a 73 kDa outer membrane protein. Functional classifications and estimated abundance levels of 790 identified proteins suggested that expression, stabilization and secretion of proteins predominate over bacterial genome replication and cell division. High relative abundances of cysteine desulphurase, serine/glycine hydroxymethyl transferase, and components of the α-ketoglutarate dehydrogenase complex in conjunction with above average abundances of glutamate dehydrogenase and proline utilization protein A support Wolbachia genome-based predictions for amino acid metabolism as a primary energy source. wStr expresses 15 Vir proteins of a Type IV secretion system and its transcriptional regulator. Proteomic characterization of a robust insect-associated Wolbachia strain provides baseline information that will inform further development of in vitro protocols for Wolbachia manipulation. PMID:25155417

  2. Effect of irradiation on cell transcriptome and proteome of rat submandibular salivary glands.

    Directory of Open Access Journals (Sweden)

    Raluca Stiubea-Cohen

    Full Text Available Salivary glands (SGs are irreversibly damaged by irradiation (IR treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG tissue.We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene.Tissue damage was seen by confocal immunofluorescence of α-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results.This data indicates that IR damages the SSG cells' ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell-cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology.

  3. Proteomic analysis of barley cell nuclei purified by flow sorting

    Czech Academy of Sciences Publication Activity Database

    Petrovská, Beáta; Jeřábková, Hana; Chamrád, I.; Vrána, Jan; Lenobel, R.; Uřinovská, J.; Šebela, M.; Doležel, Jaroslav

    2014-01-01

    Roč. 143, 1-3 (2014), s. 78-86. ISSN 1424-8581 R&D Projects: GA ČR GBP501/12/G090; GA ČR(CZ) GA14-28443S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Cell cycle * Chromatin * Flow cytometry Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.561, year: 2014 http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=MEDLINE&DestLinkType=FullRecord&UT=25059295

  4. Proteomics analysis reveals a Th17-prone cell population in presymptomatic graft-versus-host disease

    Science.gov (United States)

    Li, Wei; Liu, Liangyi; Gomez, Aurelie; Zhang, Jilu; Ramadan, Abdulraouf; Zhang, Qing; Choi, Sung W.; Zhang, Peng; Greenson, Joel K.; Liu, Chen; Jiang, Di; Virts, Elizabeth; Kelich, Stephanie L.; Chu, Hong Wei; Flynn, Ryan; Blazar, Bruce R.; Hanenberg, Helmut; Hanash, Samir; Paczesny, Sophie

    2016-01-01

    Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA– transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT. PMID:27195312

  5. Single Cell Proteomics with Ultra-High Sensitivity Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Frank, M

    2005-02-16

    This project was a joint LDRD project between PAT, CMS and NAI with the objective to develop an instrument that analyzes the biochemical composition of single cells in real-time using bioaerosol mass spectrometry (BAMS) combined with advanced laser desorption and ionization techniques. Applications include both biological defense, fundamental cell biology and biomedical research. BAMS analyzes the biochemical composition of single, micrometer-sized particles (such as bacterial cells or spores) that can be directly sampled from air or a suspension. BAMS is based on an earlier development of aerosol time of flight mass spectrometry (ATOFMS) by members of our collaboration [1,2]. Briefly, in ATOFMS and BAMS aerosol particles are sucked directly from the atmosphere into vacuum through a series of small orifices. As the particles approach the ion source region of the mass spectrometer, they cross and scatter light from two CW laser beams separated by a known distance. The timing of the two bursts of scattered light created by each ''tracked'' particle reveals the speed, location and size of the particle. This information then enables the firing of a high-intensity laser such that the resulting laser pulse desorbs and ionizes molecules from the tracked particle just as it reaches the center of the ion source region. The full spectrum of ions is then measured using a time-of-flight mass spectrometer. The ability to rapidly analyze individual particles is clearly applicable to the rapid detection of aerosolized biological warfare agents so long as agent particles can be made to produce mass spectra that are distinct from the spectra of harmless background particles. The pattern of ions formed is determined by the properties of the laser pulse, the particle, and, in aerosol matrix-assisted laser desorption/ionization (MALDI), also the MALDI matrix used. As a result, it is critical that the properties of the laser pulses used for desorption and ionization

  6. Transcriptomic and proteomic analysis of human hepatic stellate cells treated with natural taurine.

    Science.gov (United States)

    Liang, Jian; Deng, Xin; Wu, Fa-Sheng; Tang, Yan-Fang

    2013-05-01

    The aim of this study was to investigate the differential expression of genes and proteins between natural taurine (NTau)‑treated hepatic stellate cells (HSCs) and control cells as well as the underlying mechanism of NTau in inhibiting hepatic fibrosis. A microculture tetrazolium (MTT) assay was used to analyze the proliferation of NTau‑treated HSCs. Flow cytometry was performed to compare the apoptosis rate between NTau-treated and non‑treated HSCs. Proteomic analysis using a combination of 2-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) was conducted to identify the differentially expressed proteins. Microarray analysis was performed to investigate the differential expression of genes and real-time polymerase chain reaction (PCR) was used to validate the results. The experimental findings obtained demonstrated that NTau decreased HSC proliferation, resulting in an increased number of cells in the G0/G1 phase and a reduced number of cells in the S phase. Flow cytometric analysis showed that NTau-treated HSCs had a significantly increased rate of apoptosis when compared with the non‑treated control group. A total of 15 differentially expressed proteins and 658 differentially expressed genes were identified by 2DE and MS, and microarray analysis, respectively. Gene ontology (GO) functional analysis indicated that these genes and proteins were enriched in the function clusters and pathways related to cell proliferation, cellular apoptosis and oxidation. The transcriptome and proteome analyses of NTau-treated HSCs demonstrated that NTau is able to significantly inhibit cell proliferation and promote cell apoptosis, highlighting its potential therapeutic benefits in the treatment of hepatic fibrosis. PMID:23525364

  7. Effects of altered gravity on the cell cycle, actin cytoskeleton and proteome in Physarum polycephalum

    Science.gov (United States)

    He, Jie; Zhang, Xiaoxian; Gao, Yong; Li, Shuijie; Sun, Yeqing

    Some researchers suggest that the changes of cell cycle under the effect of microgravity may be associated with many serious adverse physiological changes. In the search for underlying mechanisms and possible new countermeasures, we used the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony to study the effects of altered gravity on the cell cycle, actin cytoskeleton and proteome. In parallel, the cell cycle was analyzed in Physarum incubated (1) in altered gravity for 20 h, (2) in altered gravity for 40 h, (3) in altered gravity for 80 h, and (4) in ground controls. The cell cycle, the actin cytoskeleton, and proteome in the altered gravity and ground controls were examined. The results indicated that the duration of the G2 phase was lengthened 20 min in high aspect ratio vessel (HARV) for 20 h, and prolonged 2 h in altered gravity either for 40 h or for 80 h, whereas the duration of other phases in the cell cycle was unchanged with respect to the control. The microfilaments in G2 phase had a reduced number of fibers and a unique abnormal morphology in altered gravity for 40 h, whereas the microfilaments in other phases of cell cycle were unchanged when compared to controls. Employing classical two-dimensional electrophoresis (2-DE), we examined the effect of the altered gravity on P. polycephalum proteins. The increase in the duration of G2 phase in altered gravity for 40 h was accompanied by changes in the 2-DE protein profiles, over controls. Out of a total of 200 protein spots investigated in G2 phase, which were reproducible in repeated experiments, 72 protein spots were visually identified as specially expressed, and 11 proteins were up-regulated by 2-fold and 28 proteins were down-regulated by 2-fold over controls. Out of a total of three low-expressed proteins in G2 phase in altered gravity for 40 h, two proteins were unknown proteins, and one protein was spherulin 3b by MALDI-TOF mass spectrometry (MS

  8. Proteome Profiling in Lung Injury after Hematopoietic Stem Cell Transplantation.

    Science.gov (United States)

    Bhargava, Maneesh; Viken, Kevin J; Dey, Sanjoy; Steinbach, Michael S; Wu, Baolin; Jagtap, Pratik D; Higgins, LeeAnn; Panoskaltsis-Mortari, Angela; Weisdorf, Daniel J; Kumar, Vipin; Arora, Mukta; Bitterman, Peter B; Ingbar, David H; Wendt, Chris H

    2016-08-01

    Pulmonary complications due to infection and idiopathic pneumonia syndrome (IPS), a noninfectious lung injury in hematopoietic stem cell transplant (HSCT) recipients, are frequent causes of transplantation-related mortality and morbidity. Our objective was to characterize the global bronchoalveolar lavage fluid (BALF) protein expression of IPS to identify proteins and pathways that differentiate IPS from infectious lung injury after HSCT. We studied 30 BALF samples from patients who developed lung injury within 180 days of HSCT or cellular therapy transfusion (natural killer cell transfusion). Adult subjects were classified as having IPS or infectious lung injury by the criteria outlined in the 2011 American Thoracic Society statement. BALF was depleted of hemoglobin and 14 high-abundance proteins, treated with trypsin, and labeled with isobaric tagging for relative and absolute quantification (iTRAQ) 8-plex reagent for two-dimensional capillary liquid chromatography (LC) and data dependent peptide tandem mass spectrometry (MS) on an Orbitrap Velos system in higher-energy collision-induced dissociation activation mode. Protein identification employed a target-decoy strategy using ProteinPilot within Galaxy P. The relative protein abundance was determined with reference to a global internal standard consisting of pooled BALF from patients with respiratory failure and no history of HSCT. A variance weighted t-test controlling for a false discovery rate of ≤5% was used to identify proteins that showed differential expression between IPS and infectious lung injury. The biological relevance of these proteins was determined by using gene ontology enrichment analysis and Ingenuity Pathway Analysis. We characterized 12 IPS and 18 infectious lung injury BALF samples. In the 5 iTRAQ LC-MS/MS experiments 845, 735, 532, 615, and 594 proteins were identified for a total of 1125 unique proteins and 368 common proteins across all 5 LC-MS/MS experiments. When comparing IPS to

  9. Proteomic response analysis of endothelial cells of human coronary artery to stimulation with carbachol

    Institute of Scientific and Technical Information of China (English)

    Min YU; Dong-mei CHEN; Gang HU; Hai WANG

    2004-01-01

    AIM: To identify the molecular basis of the endothelial target for acetylcholine (ETA). METHODS: Proteomic methods were used to monitor changes in protein expression in the first 10 h following the stimulation of human coronary endothelial cells with carbachol 100 μmol/L. Thirty proteins showing the largest changes were identified by mass spectrometry. RESULTS: Based on analysis with Image Master 2-D Elite software, about 623 protein spots were detected in control cells and 825 protein spots in carbachol-treated cells, the matching rate was 68.1%.Among all the detected spots, 39 were up-regulated and 29 were down-regulated, showing detectable changes varied from 1.7-3.8 folds. Forty one spots in the peptide mass fingerprints were successfully obtained. The most interesting feature was that all the four newly synthesized proteins belonged to the heat shock protein family.CONCLUSION: These identified proteins played key roles in the molecular mechanism of ETA.

  10. A proteomic study of the regulatory role for STAT-1 in cytokine-induced beta-cell death

    DEFF Research Database (Denmark)

    Rondas, Dieter; Gudmundsdottir, Valborg; D’Hertog, Wannes; Crèvecoeur, Inne; Waelkens, Etienne; Brunak, Søren; Mathieu, Chantal; Overbergh, Lut

    PURPOSE: Signal transducer and activator of transcription 1 (STAT-1) plays a crucial role in cytokine-induced beta-cell destruction. However, its precise downstream pathways have not been completely clarified. We performed a proteome analysis of cytokine-exposed C57Bl/6 and STAT-1-/- mouse islets...... assigned to small ubiquitin-related modifier 4 (SUMO4). CONCLUSIONS AND CLINICAL RELEVANCE: These findings confirm a central role for STAT-1 in pancreatic islet inflammation induced destruction and most importantly elucidate the underlying proteomic pathways involved.......PURPOSE: Signal transducer and activator of transcription 1 (STAT-1) plays a crucial role in cytokine-induced beta-cell destruction. However, its precise downstream pathways have not been completely clarified. We performed a proteome analysis of cytokine-exposed C57Bl/6 and STAT-1-/- mouse islets...

  11. Proteins upregulated by mild and severe hypoxia in squamous cell carcinomas in vitro identified by proteomics

    International Nuclear Information System (INIS)

    Background: Solid malignant tumours are characterised by an inadequate vascular system, which can give rise to micro-regional hypoxic areas. As the negative impact of tumour hypoxia is believed largely to depend on dynamic changes in gene expression, it is important to identify the genes regulated by hypoxia to further enlighten the biology behind the cellular response to hypoxia. Previous studies have demonstrated that hypoxia has an impact not only on the gene transcription, but also on gene-specific mRNA translation. Therefore, proteomics is a suitable approach to understand the complexity of gene regulation under hypoxia at protein level. In this in vitro study we have studied the proteome of cells under intermediate hypoxia (1% O2) and anoxia and compared these to normoxic (21% O2) cells to identify proteins upregulated by mild and severe hypoxia. Materials and methods: A human cervix cancer cell line (SiHa) and a human head and neck cancer cell line (FaDuDD) were used. Total cell lysate from hypoxic and normoxic cells was separated by 2-dimensional gel electrophoresis, and images were analysed using Quantity One software. Proteins from significant spots (difference in intensity by more than a factor 2) were identified by Liquid chromatography-mass spectrometry (LC-MS/MS). In order to confirm the hypoxic regulation of the identified proteins, immunoblotting and qPCR were employed when possible. Results: All together 32 spots were found to be upregulated in the hypoxic gels. Of these, 11 different proteins were successfully identified and largely confirmed by Western blotting and qPCR. Amongst these proteins are protein disulfide isomerase family A, member 6 (PDIA6) and dynein light chain roadblock-type 1 (DynLRB1). Both 2D gels and Western blots revealed that PDAI6 exhibited a cell line specific pattern; in FaDuDD there was upregulation at 1% and further upregulated at 0% compared to atmospheric air, whereas there was no upregulation in SiHa cells. DynLRB1 was

  12. Microfluidics-Based Single-Cell Functional Proteomics for Fundamental and Applied Biomedical Applications

    Science.gov (United States)

    Yu, Jing; Zhou, Jing; Sutherland, Alex; Wei, Wei; Shin, Young Shik; Xue, Min; Heath, James R.

    2014-06-01

    We review an emerging microfluidics-based toolkit for single-cell functional proteomics. Functional proteins include, but are not limited to, the secreted signaling proteins that can reflect the biological behaviors of immune cells or the intracellular phosphoproteins associated with growth factor-stimulated signaling networks. Advantages of the microfluidics platforms are multiple. First, 20 or more functional proteins may be assayed simultaneously from statistical numbers of single cells. Second, cell behaviors (e.g., motility) may be correlated with protein assays. Third, extensions to quantized cell populations can permit measurements of cell-cell interactions. Fourth, rare cells can be functionally identified and then separated for further analysis or culturing. Finally, certain assay types can provide a conduit between biology and the physicochemical laws. We discuss the history and challenges of the field then review design concepts and uses of the microchip platforms that have been reported, with an eye toward biomedical applications. We then look to the future of the field.

  13. Evaluation of preparation methods for MS-based analysis of intestinal epithelial cell proteomes

    DEFF Research Database (Denmark)

    Hesselager, Marianne Overgaard; Codrea, Marius Cosmin; Bendixen, Emøke

    2015-01-01

    The gut epithelium formed between an organism and the environment plays an essential role in host–microbe interactions, yet remains one of the least characterized mammalian tissues. Especially the membrane proteins, which are critical to bacterial adhesion, are understudied, because these proteins...... are low in abundance, and large amounts of sample is needed for their preparation and for undertaking MS-based analysis. The aim of this study was to evaluate three different methods for isolation and preparation of pig intestinal epithelial cells for MS-based analysis of the proteome. Samples were...... of ease and speed of sample preparation, as well as protein recovery. In comparison, more gentle methods where intestinal epithelial cells are harvested by shaking are more time consuming, result in lower protein yield, and are prone to increased technical variation due to multiple steps involved....

  14. Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest

    DEFF Research Database (Denmark)

    Sylvestersen, Kathrine B; Horn, Heiko; Jungmichel, Stephanie;

    2014-01-01

    contain regulated functions on their own. Collectively, we present a site-specific MMA dataset in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase....... mono-methylation (MMA) sites. We thereby identify 1,027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization......, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared to the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers...

  15. Two-dimensional gel electrophoresis analysis of the proteomes expressed in the human hepatoma cell line BEL-7404 and normal liver cell line L-02

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Proteome analysis technology has been used extensively in conducting discovery research of biology and has become one of the most essential technologies in functional genomics. The proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 have been separated by high resolution two-dimensional gel electrophoresis (2-DE) with immobilized pH gradient isoelectric focusing (IPG-IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension (IPG-DALT). The resulting images have been analyzed using 2-D analysis software. Quantitative analysis reveals that 7 protein spots are detected only in hepatoma BEL-7404 cells, 14 only in L-02 cells, and 78 protein spots show significant fluctuation in quantity in both cell lines (P<0.01).These protein spots have been displayed on a proteome differential expression map. Analysis for the reproducibility of 2-DE indicates that the positional variability in the IEF dimension is 0.73 mm, while the variability in the SDS-PAGE dimension is 0.44 mm, and the quantitative variability is 17.6%-19.2%. These results suggest that the reproducibility of 2-DE has been suitable for the study of differential expression of proteomes. Proteome differential expression maps can be useful tools for disease diagnosis, drug-target validation analysis and biological process elucidation.

  16. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

    Science.gov (United States)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  17. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  18. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  19. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

    DEFF Research Database (Denmark)

    Soufi, Boumediene; Kumar, C.; Gnad, F.;

    2010-01-01

    We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic for...... most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

  20. Organellar proteome analyses of ricin toxin-treated HeLa cells.

    Science.gov (United States)

    Liao, Peng; Li, Yunhu; Li, Hongyang; Liu, Wensen

    2016-07-01

    Apoptosis triggered by ricin toxin (RT) has previously been associated with certain cellular organellar compartments, but the diversity in the composition of the organellar proteins remains unclear. Here, we applied a shotgun proteomics strategy to examine the differential expression of proteins in the mitochondria, nuclei, and cytoplasm of HeLa cells treated and not treated with RT. Data were combined with a global bioinformatics analysis and experimental confirmations. A total of 3107 proteins were identified. Bioinformatics predictors (Proteome Analyst, WoLF PSORT, TargetP, MitoPred, Nucleo, MultiLoc, and k-nearest neighbor) and a Bayesian model that integrated these predictors were used to predict the locations of 1349 distinct organellar proteins. Our data indicate that the Bayesian model was more efficient than the individual implementation of these predictors. Additionally, a Biomolecular Interaction Network (BIN) analysis was used to identify 149 BIN subnetworks. Our experimental confirmations indicate that certain apoptosis-related proteins (e.g. cytochrome c, enolase, lamin B, Bax, and Drp1) were found to be translocated and had variable expression levels. These results provide new insights for the systematic understanding of RT-induced apoptosis responses. PMID:25227225

  1. Differential proteome analysis of bone marrow mesenchymal stem cells from adolescent idiopathic scoliosis patients.

    Directory of Open Access Journals (Sweden)

    Qianyu Zhuang

    Full Text Available Adolescent idiopathic scoliosis (AIS is a complex three-dimensional deformity of the spine. The cause and pathogenesis of scoliosis and the accompanying generalized osteopenia remain unclear despite decades of extensive research. In this study, we utilized two-dimensional fluorescence difference gel electrophoresis (2D-DIGE coupled with mass spectrometry (MS to analyze the differential proteome of bone marrow mesenchymal stem cells (BM-MSCs from AIS patients. In total, 41 significantly altered protein spots were detected, of which 34 spots were identified by MALDI-TOF/TOF analysis and found to represent 25 distinct gene products. Among these proteins, five related to bone growth and development, including pyruvate kinase M2, annexin A2, heat shock 27 kDa protein, γ-actin, and β-actin, were found to be dysregulated and therefore selected for further validation by Western blot analysis. At the protein level, our results supported the previous hypothesis that decreased osteogenic differentiation ability of MSCs is one of the mechanisms leading to osteopenia in AIS. In summary, we analyzed the differential BM-MSCs proteome of AIS patients for the first time, which may help to elucidate the underlying molecular mechanisms of bone loss in AIS and also increase understanding of the etiology and pathogenesis of AIS.

  2. Proteomic analysis of mesenchymal stem cells from normal and deep carious dental pulp.

    Directory of Open Access Journals (Sweden)

    Dandan Ma

    Full Text Available Dental pulp stem cells (DPSCs, precursor cells of odontoblasts, are ideal seed cells for tooth tissue engineering and regeneration. Our previous study has demonstrated that stem cells exist in dental pulp with deep caries and are called carious dental pulp stem cells (CDPSCs. The results indicated that CDPSCs had a higher proliferative and stronger osteogenic differentiation potential than DPSCs. However, the molecular mechanisms responsible for the biological differences between DPSCs and CDPSCs are poorly understood. The aim of this study was to define the molecular features of DPSCs and CDPSCs by comparing the proteomic profiles using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS. Our results revealed that there were 18 protein spots differentially expressed between DPSCs and CDPSCs in a narrow pH range of 4 to 7. These differently expressed proteins are mostly involved in the regulation of cell proliferation, differentiation, cell cytoskeleton and motility. In addition, our results suggested that CDPSCs had a higher expression of antioxidative proteins that might protect CDPSCs from oxidative stress. This study explores some potential proteins responsible for the biological differences between DPSCs and CDPSCs and expands our understanding on the molecular mechanisms of mineralization of DPSCs in the formation of the dentin-pulp complex.

  3. Transcriptome and proteome characterization of surface ectoderm cells differentiated from human iPSCs.

    Science.gov (United States)

    Qu, Ying; Zhou, Bo; Yang, Wei; Han, Bingchen; Yu-Rice, Yi; Gao, Bowen; Johnson, Jeffery; Svendsen, Clive N; Freeman, Michael R; Giuliano, Armando E; Sareen, Dhruv; Cui, Xiaojiang

    2016-01-01

    Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye, and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the γ-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGFβ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGFβ-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis, and development of regenerative medicine approaches. PMID:27550649

  4. The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements

    Science.gov (United States)

    Procaccini, Claudio; Carbone, Fortunata; Di Silvestre, Dario; Brambilla, Francesca; De Rosa, Veronica; Galgani, Mario; Faicchia, Deriggio; Marone, Gianni; Tramontano, Donatella; Corona, Marco; Alviggi, Carlo; Porcellini, Antonio; La Cava, Antonio; Mauri, Pierluigi; Matarese, Giuseppe

    2016-01-01

    Summary Human CD4+CD25hiFoxp3+CD127− Treg and CD4+CD25−Foxp3− Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells. PMID:26885861

  5. The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements.

    Science.gov (United States)

    Procaccini, Claudio; Carbone, Fortunata; Di Silvestre, Dario; Brambilla, Francesca; De Rosa, Veronica; Galgani, Mario; Faicchia, Deriggio; Marone, Gianni; Tramontano, Donatella; Corona, Marco; Alviggi, Carlo; Porcellini, Antonio; La Cava, Antonio; Mauri, Pierluigi; Matarese, Giuseppe

    2016-02-16

    Human CD4(+)CD25(hi)Foxp3(+)CD127(-) Treg and CD4(+)CD25(-)Foxp3(-) Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells. PMID:26885861

  6. Proteomics Technologies and Challenges

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proteomics is the study of proteins and their interactions in a cell. With the completion of the Human Genome Project, the emphasis is shifting to the protein compliment of the human organism. Because proteome reflects more accurately on the dynamic state of a cell, tissue, or organism, much is expected from proteomics to yield better disease markers for diagnosis and therapy monitoring. The advent of proteomics technologies for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of diseases. High-throughput proteomics technologies combining with advanced bioinformatics are extensively used to identify molecular signatures of diseases based on protein pathways and signaling cascades. Mass spectrometry plays a vital role in proteomics and has become an indispensable tool for molecular and cellular biology. While the potential is great, many challenges and issues remain to be solved, such as mining low abundant proteins and integration of proteomics with genomics and metabolomics data. Nevertheless, proteomics is the foundation for constructing and extracting useful knowledge to biomedical research. In this review, a snapshot of contemporary issues in proteomics technologies is discussed.

  7. A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling.

    Science.gov (United States)

    Ao, Jie; Aldabbous, Mash'el; Notaro, Marysa J; Lojacono, Mark; Free, Stephen J

    2016-09-01

    A proteomic analysis of the conidial cell wall identified 35 cell wall proteins. A comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared, and that these shared cell wall proteins were cell wall biosynthetic proteins or cell wall structural proteins. Deletion mutants for 34 of the genes were analyzed for phenotypes indicative of conidial cell wall defects. Mutants for two cell wall glycosyl hydrolases, the CGL-1 β-1,3-glucanase (NCU07523) and the NAG-1 exochitinase (NCU10852), were found to have a conidial separation phenotype. These two enzymes function in remodeling the cell wall between adjacent conidia to facilitate conidia formation and dissemination. Using promoter::RFP and promoter::GFP constructs, we demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided for conidia-specific gene expression or for a significant increase in their expression during conidiation. PMID:27381444

  8. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Holger A. Russ

    2016-01-01

    Full Text Available Current approaches in human embryonic stem cell (hESC to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1, Neuroplastin (NPTN, and the Laminin α-2 subunit (LAMA2. Two of the three factors (LGALS1 and LAMA2 increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

  9. Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

    Science.gov (United States)

    Russ, Holger A; Landsman, Limor; Moss, Christopher L; Higdon, Roger; Greer, Renee L; Kaihara, Kelly; Salamon, Randy; Kolker, Eugene; Hebrok, Matthias

    2016-01-01

    Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation. PMID:26681951

  10. Proteome characterization of sea star coelomocytes--the innate immune effector cells of echinoderms.

    Science.gov (United States)

    Franco, Catarina F; Santos, Romana; Coelho, Ana V

    2011-09-01

    Sea star coelomic fluid is in contact with all internal organs, carrying signaling molecules and a large population of circulating cells, the coelomocytes. These cells, also known as echinoderm blood cells, are responsible for the innate immune responses and are also known to have an important role in the first stage of regeneration, i.e. wound closure, necessary to prevent disruption of the body fluid balance and to limit the invasion of pathogens. This study focuses on the proteome characterization of these multifunctional cells. The identification of 358 proteins was achieved using a combination of two techniques for protein separation (1-D SDS-PAGE followed by nanoLC and 2-D SDS-PAGE) and MALDI-TOF/TOF MS for protein identification. To our knowledge, the present report represents the first comprehensive list of sea star coelomocyte proteins, constituting an important database to validate many echinoderm-predicted proteins. Evidence for new pathways in these particular echinoderm cells are also described, and thus representing a valuable resource to stimulate future studies aiming to unravel the homology with vertebrate immune cells and particularly the origins of the immune system itself. PMID:21751360

  11. Proteomic analysis of apoptosis induction by lariciresinol in human HepG2 cells.

    Science.gov (United States)

    Ma, Zhan-Jun; Wang, Xue-Xi; Su, Gang; Yang, Jing-Jing; Zhu, Ya-Juan; Wu, You-Wei; Li, Jing; Lu, Li; Zeng, Long; Pei, Hai-Xia

    2016-08-25

    Lariciresinol (LA) is a traditional Chinese medicine possessing anticancer activity, but its mechanism of action remains unclear. The present study explored the effects of LA on human HepG2 cells and the underlying mechanism. Our data indicated that LA inhibited cell proliferation and induced cell cycle arrest in S phase, subsequently resulting in apoptosis in HepG2 cells. Using a proteomics approach, eight differentially expressed proteins were identified. Among them, three proteins, glyceraldehyde-3-phosphate, UDP-glucose 4-epimerase, and annexin A1, were upregulated, while the other five proteins, heat shock protein 27, haptoglobin, tropomodulin-2, tubulin alpha-1A chain, and brain acid soluble protein 1, were downregulated; all of these proteins are involved in cell proliferation, metabolism, cytoskeletal organization, and movement. Network analysis of these proteins suggested that the ubiquitin-conjugating enzyme (UBC) plays an important role in the mechanism of LA. Western blotting confirmed downregulation of heat shock protein 27 and upregulation of ubiquitin and UBC expression levels in LA-treated cells, consistent with the results of two-dimensional electrophoresis and a STRING software-based analysis. Overall, LA is a multi-target compound with anti-cancer effects potentially related to the ubiquitin-proteasome pathway. This study will increase our understanding of the anticancer mechanisms of LA. PMID:27417256

  12. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

    DEFF Research Database (Denmark)

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T;

    2009-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research...

  13. Non-biased enrichment does not improve quantitative proteomic delineation of reovirus T3D-infected HeLa cell protein alterations

    OpenAIRE

    KevinM.Coombs

    2012-01-01

    Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy st...

  14. High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells.

    Directory of Open Access Journals (Sweden)

    Han-Chen Chiu

    Full Text Available Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC in combination with high-throughput mass spectrometry (MS. Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

  15. Proteomic analysis of the effect of iptakalim on human pulmonary arterial smooth muscle cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Mingxia YANG; Zhengxia LIU; Shu ZHANG; Yu JING; Shijiang ZHANG; Weiping XIE; Lei MA; Changliang ZHU; Hong WANG

    2009-01-01

    Aim:To investigate the anti-proliferative effect of iptakalim (Ipt),a newly selective KATP channel opener,in endothelin-1 (ET-1)-induced human pulmonary arterial smooth muscle cells (PASMCs) using proteomic analysis.Methods: Human PASMCs were incubated with ET-1 (10-8 mol/L) and ETA (10-8 mol/L) plus iptaklim (10-5 mol/L) for 24 h.Analysis via 2-DE gel electrophoresis and MALDI-TOF-MS was employed to display the different protein profiles of whole-cell protein from cultures of control,ET-1 treatment alone,and treatment with ET-1 and iptaklim combined.Real time RT-PCR and Western blot analysis were used to confirm the proteomic analysis.Results: When iptakalim inhibited the proliferative effect of ET-1 in human PASMCs by opening the KATP channels,the expression of different groups of cellular proteins was changed,including cytoskeleton-associated proteins,plasma mem-brane proteins and receptors,chaperone proteins,ion transport-associated proteins,and glycolytic and metabolism-associ-ated proteins.We found that iptakalim could inhibit the proliferation of human PASMCs partly by affecting the expression of Hsp60,vimentin,nucleoporin P54 (NUP54) and Bcl-XL by opening the KATP channel.Conclusion: The data suggest that a wide range of signaling pathways may be involved in abolishing ET-1-induced prolif-eration of human PASMCs following iptakalim treatment.

  16. Proteomic analysis of hippocampal dentate granule cells in frontotemporal lobar degeneration: Application of laser capture technology.

    Directory of Open Access Journals (Sweden)

    YairM.Gozal

    2011-04-01

    Full Text Available Frontotemporal lobar degeneration (FTLD is the most common cause of dementia with pre-senile onset, accounting for as many as 20% of cases. A common subset of FTLD cases is characterized by the presence of ubiquitinated inclusions in vulnerable neurons (FTLD-U. While the pathophysiological mechanisms underlying neurodegeneration in FTLD-U have not yet been elucidated, the presence of inclusions in this disease indicates enhanced aggregation of one or several proteins. Moreover, these inclusions suggest altered expression, processing, or degradation of proteins during FTLD-U pathogenesis. Thus, one approach to understanding disease mechanisms is to delineate the molecular changes in protein composition in FTLD-U brain. Using a combined approach consisting of laser capture microdissection (LCM and high resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS, we identified 1252 proteins in hippocampal dentate granule cells excised from three post-mortem FTLD-U and three unaffected control cases processed in parallel. Additionally, we employed a labeling-free quantification technique to compare the abundance of the identified proteins between FTLD-U and control cases. Quantification revealed 54 proteins with selective enrichment in FTLD-U, including TAR DNA binding protein 43 (TDP-43, a recently identified component of ubiquitinated inclusions. Moreover, 19 proteins were selectively decreased in FTLD-U. Subsequent immunohistochemical analysis of TDP-43 and three additional protein candidates suggests that our proteomic profiling of FTLD-U dentate granule cells reveals both inclusion-associated proteins and non-aggregated disease-specific proteins. Application of LCM is a valuable tool in the molecular analysis of complex tissues, and its application in the proteomic characterization of neurodegenerative disorders such as FTLD-U may be used to identify proteins altered in disease.

  17. Proteomic biomarkers of peripheral blood mononuclear cells obtained from postmenopausal women undergoing an intervention with soy isoflavones

    OpenAIRE

    Fuchs, D; Vafeiadou, K.; Hall, W.L.; Daniel, H; Williams, C.M.; Schroot, J.H.; Wenzel, U.

    2007-01-01

    Background: The incidence of cardiovascular diseases increases after menopause, and soy consumption is suggested to inhibit disease development. Objective: The objective was to identify biomarkers of response to a dietary supplementation with an isoflavone extract in postmenopausal women by proteome analysis of peripheral blood mononuclear cells. Design: The study with healthy postmenopausal woman was performed in a placebo-controlled sequential design. Peripheral mononuclear blood cells were...

  18. Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells

    OpenAIRE

    Kuster Niels; Nylund Reetta; Leszczynski Dariusz

    2010-01-01

    Abstract Background Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in exp...

  19. Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells

    OpenAIRE

    Nylund, Reetta; KUSTER, Niels; Leszczynski, Dariusz

    2010-01-01

    Background Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression o...

  20. Proteomics analysis of human umbilical vein endothelial cells (HUVEC) after treatment with low molecular weight heparin

    Institute of Scientific and Technical Information of China (English)

    YanPAN; Jun-huaWANG; He-mingYU; Xue-junLI

    2004-01-01

    AIM: The endothelium is involved in the generation and the regulation of multiple physiological and pathological processes of blood vessels. Previously we confirmed low molecular weight heparin (LMWH) could inhibit tumor metastasis by protecting human umbilical vein endothelial cells (HUVEC). To understand the effects of LMWH on the protein expression of HUVEC, we performed a comprehensive proteomics to survey global changes in proteins after LMWH treatment in HUVEC cells. METHODS:

  1. Proteomic identification of alpha-2-HS-glycoprotein as a plasma biomarker of hypopharyngeal squamous cell carcinoma

    OpenAIRE

    TIAN, WEN-DONG; Li, Jun-Zheng; Hu, Shui-Wang; Peng, Xiao-wei; Li, Gang; LIU, Xiong; Chen, Huai-hong; Xu, Xia; Li, Xiang-ping

    2015-01-01

    Hypopharyngeal squamous cell carcinoma (HSCC) has very poor prognosis compared with other head and neck squamous cell carcinomas. Late-stage diagnosis of HSCC increases mortality. Therefore, more effective biomarkers for early diagnosis of HSCC are necessary. Unfortunately, appropriate biomarkers for clinical diagnosis and prognosis have not been identified yet. However, recent progresses in quantitative proteomics have offered opportunities to identify plasma proteins as biomarkers for HSCC....

  2. Genomic and proteomic analyses of Prdm5 reveal interactions with insulator binding proteins in embryonic stem cells

    DEFF Research Database (Denmark)

    Galli, Giorgio Giacomo; Carrara, Matteo; Francavilla, Chiara;

    2013-01-01

    find that Prdm5 is highly expressed in mouse embryonic stem cells (mES) and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next generation sequencing technologies we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that......-occupies genomic loci. In summary, our data indicate how Prdm5 may modulate transcription by interacting with factors involved in genome organization in mouse embryonic stem cells....

  3. iTRAQ-based proteomic analysis of dioscin on human HCT-116 colon cancer cells.

    Science.gov (United States)

    Chen, Hao; Xu, Lina; Yin, Lianhong; Xu, Youwei; Han, Xu; Qi, Yan; Zhao, Yanyan; Liu, Kexin; Peng, Jinyong

    2014-01-01

    Dioscin shows various pharmacological effects. However, its activity on colorectal cancer is still unknown. The present work showed that dioscin significantly inhibited cell proliferation on human HCT-116 colon cancer cells, and affected Ca(2+) release and ROS generation. The content of nitric oxide (NO) and its producer inducible NO synthase (iNOS) associated with DNA damage and aberrant cell signaling were assayed using the kits. DNA damage and cell apoptosis caused by dioscin were also analyzed through single-cell gel electrophoresis and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling assays. The results showed that dioscin increased the levels of NO and inducible NO synthase. The comet length in dioscin-treated groups was much longer than that of the control group, and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells (apoptotic cells) was significantly increased by the compound (p dioscin caused mitochondrial damage and G2/M cell cycle arrest through transmission electron microscopy and flow cytometry analysis, respectively. To study the cytotoxic mechanism of dioscin, an iTRAQ-based proteomics approach was used. There were 288 significantly different proteins expressed in response to dioscin, which were connected with each other and were involved in different Kyoto Encyclopedia of Genes and Genomes pathways. Then, some differentially expressed proteins involved in oxidative phosphorylation, Wnt, p53, and calcium signaling pathways were validated by Western blotting and quantitative real-time PCR assays. Our work elucidates the molecular mechanism of dioscin-induced cytotoxicity in colon cancer cells, and the identified targets may be useful for treatment of colorectal cancer in future. PMID:24420967

  4. Comparative Studies of the Proteome, Glycoproteome, and N-Glycome of Clear Cell Renal Cell Carcinoma Plasma before and after Curative Nephrectomy

    OpenAIRE

    Gbormittah, Francisca O.; Lee, Ling Y.; Taylor, KyOnese; Hancock, William S.; Iliopoulos, Othon

    2014-01-01

    Clear cell renal cell carcinoma is the most prevalent of all reported kidney cancer cases, and currently there are no markers for early diagnosis. This has stimulated great research interest recently because early detection of the disease can significantly improve the low survival rate. Combining the proteome, glycoproteome, and N-glycome data from clear cell renal cell carcinoma plasma has the potential of identifying candidate markers for early diagnosis and prognosis and/or to monitor dise...

  5. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

    International Nuclear Information System (INIS)

    Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm ± 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

  6. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Atay, Safinur [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Gercel-Taylor, Cicek [Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States); Kesimer, Mehmet [Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC (United States); Taylor, Douglas D., E-mail: ddtaylor@louisville.edu [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States)

    2011-05-01

    Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

  7. Comparative proteomics analysis of lanthanum citrate complex-induced apoptosis in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In a previous study,the lanthanum citrate complex([LaCit2]3-) has been found to induce apoptosis in the human HeLa cervical cancer cell line.To clarify the mechanism,we carried out comparative proteomics analysis between treated and control cells.Differentially expressed proteins were separated electrophoretically and identified by MALDI-TOF/TOF tandem mass spectrometry.There were profound changes in 14 proteins related to mitochondrial function and oxidative stress,suggesting that mitochondrial dysfunction plays a key role in [LaCit2]3--induced apoptosis.This was confirmed by a decrease in the mitochondrial transmembrane potential,and increases in cytochrome c release and reactive oxygen species generation in [LaCit2]3--treated cells.Western blotting analyses show that [LaCit2]3--induced apoptosis was accompanied by the activation of caspase-9 and the specific proteolytic cleavage of PARP,leading to an increase in the proapoptotic protein Bax and a decrease in the antiapoptotic protein Bcl-2.These results suggest that [LaCit2]3-induced the apoptosis of HeLa cells through oxidative stress mediated pathway involving MT participation.

  8. The effects of quercetin on SW480 human colon carcinoma cells: a proteomic study

    Directory of Open Access Journals (Sweden)

    Hargrove James L

    2005-03-01

    Full Text Available Abstract Background High fruit and vegetable intake is known to reduce the risk of colon cancer. To improve understanding of this phenomenon the action of different phytochemicals on colon cells has been examined. One such compound is quercetin that belongs to the group known as flavonoids. The purpose of this study was to determine the influence of quercetin on the proteome of the SW480 human colon adenocarcinoma cell line, specifically to identify proteins that could be the molecular targets of quercetin in its amelioration of the progression of colon cancer. To this end, two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins that underwent a change in expression following treatment of the cells with 20 μM quercetin. This could elucidate how quercetin may reduce the progression of colon cancer. Results Quercetin treatment of the SW480 human colon cancer cells was found to result in the decreased expression of three proteins and the increased expression of one protein. The identified proteins with decreased expression were type II cytoskeletal 8 keratin and NADH dehydrogenase Fe-S protein 3. The other protein with decreased expression was not identified. The protein with increased expression belonged to the annexin family. Conclusion Several proteins were determined to have altered expression following treatment with quercetin. Such changes in the levels of these particular proteins could underlie the chemo-protective action of quercetin towards colon cancer.

  9. Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

    Science.gov (United States)

    Billing, Anja M; Ben Hamidane, Hisham; Dib, Shaima S; Cotton, Richard J; Bhagwat, Aditya M; Kumar, Pankaj; Hayat, Shahina; Yousri, Noha A; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

    2016-01-01

    Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. PMID:26857143

  10. Proteomics of Rice Seed Germination

    Directory of Open Access Journals (Sweden)

    Dongli eHe

    2013-07-01

    Full Text Available Seed is a condensed form of plant. Under suitable environmental conditions, it can resume the metabolic activity from physiological quiescent status, and mobilize the reserves, biosynthesize new proteins, regenerate organelles and cell membrane, eventually protrude the radicle and enter into seedling establishment. So far, how these activities are regulated in a coordinated and sequential manner is largely unknown. With the availability of more and more genome sequence information and the development of mass spectrometry (MS technology, proteomics has been widely applied in analyzing the mechanisms of different biological processes, and proved to be very powerful. Regulation of rice seed germination is critical for rice cultivation. In recent years, a lot of proteomic studies have been conducted in exploring the gene expression regulation, reserves mobilization and metabolisms reactivation, which brings us new insights on the mechanisms of metabolism regulation during this process. Nevertheless, it also invokes a lot of questions. In this mini-review, we summarized the progress in the proteomic studies of rice seed germination. The current challenges and future perspectives were also discussed, which might be helpful for the following studies.

  11. Quantitative proteomic study of human prostate cancer cells with different metastatic potentials.

    Science.gov (United States)

    Li, Qun; Li, Yilei; Wang, Yanying; Cui, Zheng; Gong, Lulu; Qu, Zhigang; Zhong, Yanping; Zhou, Jun; Zhou, Ying; Gao, Yong; Li, Yulin

    2016-04-01

    Metastatic dissemination is a feature of most cancers including prostate cancer (PCa), and is the main cause of treatment failure and mortality. The aim of the study is to explore the mechanisms of PCa metastasis and to search for potential prognostic markers using proteomics. Two-dimensional fluorescent differential gel electrophoresis (2D-DIGE) was used to quantify proteins in normal prostate epithelial cells, bone metastasis-derived PC-3 cells, and visceral metastasis-derived PC-3M cells. Metastatic potential was confirmed by flow cytometry, electron microscopy, proliferating cell nuclear antigen assay, and wound healing assay. Differential protein expression was compared between PCa cells with different metastatic potentials (LNcap, DU145, PC-3 and PC-3M) and normal prostate epithelial cells (RWPE-1). Selected candidate proteins in human prostate tissues were analyzed using GOA, UniProt and GeneCards analyses. Eighty-six proteins were differentially expressed between cell lines (>1.5-fold, P<0.05). Among them, twelve proteins were identified by MALDI-TOF-MS. One protein was upregulated in normal prostate epithelial cells, nine proteins were upregulated in PC-3, and two proteins were upregulated in PC-3M. Proteins were divided into five groups according to their functions. The SETDB1 protein was closely associated with the prognosis of PCa. Bioinformatics suggested that SETDB1 might promote PCa bone metastasis through the WNT pathway. In conclusion, SETDB1 might be associated with the development of bone metastases from PCa. Further study is necessary to assess its exact role in PCa. PMID:26846621

  12. Quantitative proteomic analysis for radiation-induced cell cycle suspension in 92-1 melanoma cell line

    International Nuclear Information System (INIS)

    Melanoma is a malignant tumor with high invasive and metastatic properties. Though radiation is the major therapy for melanoma, its radio-resistance has been shown to severely influence the clinical outcome. So it is imperative to enhance the sensitivity of uveal melanoma cells to radiotherapy. Previously, we found that the cell cycle of 92-1 uveal melanoma cells was suspended and remained unchanged for up to 5 days after exposure to 10 Gy of X-rays, which might be relevant to the high radio-sensitivity of 92-1 cells. To further investigate the cell cycle suspension-associated proteins, we employed two analyses with stable isotope labeling with amino acids in cell culture technology and two-dimensional liquid chromatography tandem mass spectrometry. Cells were incubated for 15 h or 48 h after irradiation with 10 Gy of X-rays. We identified a total of 737 proteins at 15 h (Group A) and 530 proteins at 48 h post-irradiation (Group B). The gene ontology biological pathway was used to obtain a systems level view of proteome changes in 92-1 cells under cell cycle suspension. We further selected the significantly changed proteins for investigation of their potential contribution to cell cycle suspension, growth arrest and cell senescence. These proteins are involved in the cell cycle, stress response, glycolysis and the tricarboxylic acid cycle, etc. Our study expected to reveal potential marker proteins associated with cell suspension induced by irradiation, which might contribute to understanding the mechanism beyond the cell cycle suspension. (author)

  13. Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

    Directory of Open Access Journals (Sweden)

    Qui JX

    2015-01-01

    Full Text Available Jia-Xuan Qiu,1,2 Zhi-Wei Zhou, 3,4 Zhi-Xu He,4 Ruan Jin Zhao,5 Xueji Zhang,6 Lun Yang,7 Shu-Feng Zhou,3,4 Zong-Fu Mao11School of Public Health, Wuhan University, Wuhan, Hubei, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 4Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China; 5Center for Traditional Chinese Medicine, Sarasota, FL, USA; 6Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 7Bio-X Institutes, Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders (Ministry of Education, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: Plumbagin (PLB has exhibited a potent anticancer effect in preclinical studies, but the molecular interactome remains elusive. This study aimed to compare the quantitative proteomic responses to PLB treatment in human prostate cancer PC-3 and DU145 cells using the approach of stable-isotope labeling by amino acids in cell culture (SILAC. The data were finally validated using Western blot assay. First, the bioinformatic analysis predicted that PLB could interact with 78 proteins that were involved in cell proliferation and apoptosis, immunity, and signal transduction. Our quantitative proteomic study using SILAC revealed that there were at least 1,225 and 267 proteins interacting with PLB and there were 341 and 107 signaling pathways and cellular functions potentially regulated by PLB in PC-3 and DU145 cells, respectively. These proteins and pathways played a

  14. Analysis of transcriptomic and proteomic profiles demonstrates improved Madin-Darby canine kidney cell function in a renal microfluidic biochip.

    Science.gov (United States)

    Snouber, Leila Choucha; Letourneur, Franck; Chafey, Philippe; Broussard, Cedric; Monge, Matthieu; Legallais, Cécile; Leclerc, Eric

    2012-01-01

    We have evaluated the influence of the microfluidic environment on renal cell functionality. For that purpose, we performed a time lapse transcriptomic and proteomic analysis in which we compared gene and protein expressions of Madin-Darby canine kidney cells after 24 h and 96 h of culture in both microfluidic biochips and plates. The transcriptomic and proteomic integration revealed that the ion transporters involved in calcium, phosphate, and sodium homoeostasis and several genes involved in H(+) transporters and pH regulation were up-regulated in microfluidic biochips. Concerning drug metabolism, we found Phase I (CYP P450), Phase II enzymes (GST), various multidrug resistance genes (MRP), and Phase III transporters (SLC) were also up-regulated in the biochips. Furthermore, the study shows that those inductions were correlated with the induction of the Ahr and Nrf-2 dependent pathways, which results in a global cytoprotective response induced by the microenvironment. However, there was no apoptosis situation or cell death in the biochips. Microfluidic biochips may thus provide an important insight into exploring xenobiotic injury and transport modifications in this type of bioartificial microfluidic kidney. Finally, the investigation demonstrated that combining the transcriptomic and proteomic analyses obtained from a cell "on chip" culture would provide a pertinent new tool in the mechanistic interpretation of cellular mechanisms for predicting kidney cell toxicity and renal clearance in vitro. PMID:22095740

  15. Comparative Proteomics Study on Human High-metastatic Large Cell Lung Cancer Cell Lines Before and After Transfecting with nm23-H1 Gene

    Directory of Open Access Journals (Sweden)

    Liwei GAO

    2010-10-01

    Full Text Available Background and objective As a tumor metastasis suppressor gene, the functions of nm23-H1 gene are still unclear. The aim of this study is to better understand the mechanism of lung cancer metastasis and to find new biomarkers for early diagnosis and new target for therapy by conducting comparative proteomics between the human high-metastatic large cell lung cancer cell lines (L9981 and L9981-nm23-H1 (constructed with transfecting nm23-H1 gene into the L9981 cell line. Methods The total proteins of L9981 and L9981-nm23-H1 were separated by immobilized pH gradient (IPG-based 2-dimensional electrophoresis (2-DE; the significantly differently expressed proteins were examined by mass spectrometry and analyzed by bioinformatics. Results It was observed that nm23-H1 gene transfection caused remarkable changes of the proteome of L9981 compared with L9981-nm23-H1 cells: 5 proteins were deleted, 9 proteins appeared, 16 proteins downregulated, and 12 proteins up-regulated. These proteins are involved in cell framework, signal transduction, metabolism, proliferation and metastasis. Conclusion After nm23-H1 gene is transfected into L9981, proteome in L9981 is remarkably changed. These changes of the proteome could serve as a basis for reversing the invasive and metastatic phenotype in lung cancer and elucidating the machanisms of the metastasis of lung cancer.

  16. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

    OpenAIRE

    Yang, Jing; McCormick, Mark A.; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K.; Li, Hao

    2015-01-01

    In this work, we took a proteome-centric view to analyze the cell division and lifespan asymmetry between mother and daughter cells in budding yeast. Using a flow cytometry-based, high-throughput approach, we quantified the partitioning of the proteome and identified 74 mother-enriched and 60 daughter-enriched proteins. Functional analysis of these proteins suggests mechanisms of asymmetric partitioning at an organelle/suborganelle level. We found that mother-enriched proteins are much more l...

  17. In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

    Science.gov (United States)

    Wang, Jigang; Zhang, Jianbin; Zhang, Chong-Jing; Wong, Yin Kwan; Lim, Teck Kwang; Hua, Zi-Chun; Liu, Bin; Tannenbaum, Steven R; Shen, Han-Ming; Lin, Qingsong

    2016-01-01

    To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQ(TM) quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway Analysis(TM) (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death. PMID:26915414

  18. Proteomic Identification of LASP-1 Down-regulation After RNAi Urokinase Silencing in Human Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Alessandro Salvi

    2009-02-01

    Full Text Available In human hepatocellular carcinoma (HCC, the high expression of urokinase-type plasminogen activator (uPA is an unfavorable prognostic factor and a therapeutic target. To identify the downstream effects of uPA silencing by RNA interference, we studied proteome modifications of uPA-inhibited SKHep1C3 cells, an HCC-derived cell line. The study with two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry showed Lim and SH3 protein 1 (LASP-1, cytokeratin 1 (CK-1, cytokeratin 10 (CK-10, and heterogeneous nuclear ribonucleoprotein H1 down-modulation after uPA inhibition. LASP-1, CK-1, and CK-10 are involved in cytoskeleton dynamics as heterogeneous nuclear ribonucleoprotein H1 takes part in the mRNA processing and stability. We first confirmed the proteomic data by Western blot and immunoflorescence and then explored the link between uPA and LASP-1. The ectopic expression of uPA and LASP-1 supported the proteomic results and showed that uPA up-regulation increased LASP-1 expression and that both were implicated in SKHep1C3 motility. siRNA LASP-1 inhibition showed that LASP-1 was involved in actin microfilaments organization of SKHep1C3 cells. The disruption of the actin microfilaments after LASP-1 depletion increased uPA secretion and SKHep1C3 motility. Our results would suggest the hypothesis that uPA and LASP-1 expression may be coordinated in HCC-derived cells. In summary, the proteomic identification of a set of uPA downstream proteins provides new insight into the function of uPA in HCC cells.

  19. Data from proteomic characterization of the role of Snail1 in murine mesenchymal stem cells and 3T3-L1 fibroblasts differentiation

    Directory of Open Access Journals (Sweden)

    A. Peláez-García

    2015-09-01

    Full Text Available The transcription factor (TF Snail1 is a major inducer of the epithelial–mesenchymal transition (EMT during embryonic development and cancer progression. Ectopic expression of Snail in murine mesenchymal stem cells (mMSC abrogated their differentiation to osteoblasts or adipocytes. We used either stable isotopic metabolic labeling (SILAC for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT for mMSC stably transfected cells with Snail1 or control. We carried out a proteomic analysis on the nuclear fraction since Snail is a nuclear TF that mediates its effects mainly through the regulation of other TFs. Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifiers PXD001529 and PXD002157 (Vizcaino et al., 2014 [1]. Data are associated with a research article published in Molecular and Cellular Proteomics (Pelaez-Garcia et al., 2015 [2].

  20. Polymers in cell encapsulation from an enveloped cell perspective.

    Science.gov (United States)

    de Vos, Paul; Lazarjani, Hamideh Aghajani; Poncelet, Denis; Faas, Marijke M

    2014-04-01

    In the past two decades, many polymers have been proposed for producing immunoprotective capsules. Examples include the natural polymers alginate, agarose, chitosan, cellulose, collagen, and xanthan and synthetic polymers poly(ethylene glycol), polyvinyl alcohol, polyurethane, poly(ether-sulfone), polypropylene, sodium polystyrene sulfate, and polyacrylate poly(acrylonitrile-sodium methallylsulfonate). The biocompatibility of these polymers is discussed in terms of tissue responses in both the host and matrix to accommodate the functional survival of the cells. Cells should grow and function in the polymer network as adequately as in their natural environment. This is critical when therapeutic cells from scarce cadaveric donors are considered, such as pancreatic islets. Additionally, the cell mass in capsules is discussed from the perspective of emerging new insights into the release of so-called danger-associated molecular pattern molecules by clumps of necrotic therapeutic cells. We conclude that despite two decades of intensive research, drawing conclusions about which polymer is most adequate for clinical application is still difficult. This is because of the lack of documentation on critical information, such as the composition of the polymer, the presence or absence of confounding factors that induce immune responses, toxicity to enveloped cells, and the permeability of the polymer network. Only alginate has been studied extensively and currently qualifies for application. This review also discusses critical issues that are not directly related to polymers and are not discussed in the other reviews in this issue, such as the functional performance of encapsulated cells in vivo. Physiological endocrine responses may indeed not be expected because of the many barriers that the metabolites encounter when traveling from the blood stream to the enveloped cells and back to circulation. However, despite these diffusion barriers, many studies have shown optimal

  1. Proteomic Profiling of Recombinant Escherichia coli in High-Cell- Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

    OpenAIRE

    Aldor, Ilana S.; Krawitz, Denise C.; Forrest, William; Chen, Christina; Nishihara, Julie C.; Joly, John C.; Champion, Kathleen M.

    2005-01-01

    By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein sp...

  2. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    OpenAIRE

    Gama, José B.; Steffen Ohlmeier; Martins, Teresa G.; Fraga, Alexandra G.; Belém Sampaio-Marques; Carvalho, Maria A.; Fernanda Proença; Silva, Manuel T.; Jorge Pedrosa; Paula Ludovico

    2014-01-01

    Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This reveal...

  3. Proteomic Analysis of the Action of the Mycobacterium ulcerans Toxin Mycolactone: Targeting Host Cells Cytoskeleton and Collagen

    OpenAIRE

    Gama, José B.; Ohlmeier, S.; Martins, Teresa G.; Fraga, Alexandra G.; Marques, Belém Sampaio; Carvalho, M. Alice; Proença, M. Fernanda R. P.; Silva, Manuel T.; Pedrosa, Jorge; Ludovico, Paula

    2014-01-01

    Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This reveal...

  4. Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

    KAUST Repository

    Janjanam, Jagadeesh

    2013-10-01

    Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Directory of Open Access Journals (Sweden)

    Shufeng Liu

    Full Text Available Hepatitis C virus (HCV poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP, UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1 and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection.

  6. Comparative Proteomics Reveals Important Viral-Host Interactions in HCV-Infected Human Liver Cells.

    Science.gov (United States)

    Liu, Shufeng; Zhao, Ting; Song, BenBen; Zhou, Jianhua; Wang, Tony T

    2016-01-01

    Hepatitis C virus (HCV) poses a global threat to public health. HCV envelop protein E2 is the major component on the virus envelope, which plays an important role in virus entry and morphogenesis. Here, for the first time, we affinity purified E2 complex formed in HCV-infected human hepatoma cells and conducted comparative mass spectrometric analyses. 85 cellular proteins and three viral proteins were successfully identified in three independent trials, among which alphafetoprotein (AFP), UDP-glucose: glycoprotein glucosyltransferase 1 (UGT1) and HCV NS4B were further validated as novel E2 binding partners. Subsequent functional characterization demonstrated that gene silencing of UGT1 in human hepatoma cell line Huh7.5.1 markedly decreased the production of infectious HCV, indicating a regulatory role of UGT1 in viral lifecycle. Domain mapping experiments showed that HCV E2-NS4B interaction requires the transmembrane domains of the two proteins. Altogether, our proteomics study has uncovered key viral and cellular factors that interact with E2 and provided new insights into our understanding of HCV infection. PMID:26808496

  7. Comparison of Chloroflexus aurantiacus strain J-10-fl proteomes of cells grown chemoheterotrophically and photoheterotrophically

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Li; Bryant, Donald A.; Schepmoes, Athena A.; Vogl, Kajetan; Smith, Richard D.; Lipton, Mary S.; Callister, Stephen J.

    2012-01-17

    Chloroflexus aurantiacus J-10-fl is a thermophilic green bacterium, a filamentous anoxygenic phototroph, and the model organism of the phylum Chloroflexi. We applied high-throughput, liquid chromatography-mass spectrometry in a global quantitative proteomics investigation of C. aurantiacus cells grown under oxic (chemoorganoheterotrophically) and anoxic (photoorganoheterotrophically) redox states. Our global analysis identified 13,524 high-confidence peptides that matched to 1,286 annotated proteins, 242 of which were either uniquely identified or significantly increased in abundance under anoxic culture conditions. Fifty-three of the 242 proteins are previously characterized photosynthesis-related proteins, including chlorosome proteins, proteins involved in the bacteriochlorophyll biosynthesis, 3-hydroxypropionate (3-OHP) CO2 fixation pathway, and components of electron transport chains. The remaining 190 proteins have not previously been reported. Of these, five proteins were found to be encoded by genes from a novel operon and observed only in photoheterotrophically grown cells. These proteins candidates may prove useful in further deciphering the phototrophic physiology of C. aurantiacus and other filamentous anoxygenic phototrophs.

  8. Progenitor cells in the kidney: biology and therapeutic perspectives

    NARCIS (Netherlands)

    Rookmaaker, M.B.; Verhaar, M.C.; Zonneveld, A.J. van; Rabelink, T.J.

    2004-01-01

    Progenitor cells in the kidney: Biology and therapeutic perspectives. The stem cell may be viewed as an engineer who can read the blue print and become the building. The role of this fascinating cell in physiology and pathophysiology has recently attracted a great deal of interest. The archetype of

  9. Assessing CMT cell line stability by two dimensional polyacrylamide gel electrophoresis and mass spectrometry based proteome analysis

    DEFF Research Database (Denmark)

    Zhang, Kelan; Wrzesinski, Krzysztof; Fey, Stephen J;

    2008-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high...... useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs....

  10. Comparison of functional proteomic analyses of human breast cancer cell lines T47D and MCF7.

    Directory of Open Access Journals (Sweden)

    Juliette Adjo Aka

    Full Text Available T47D and MCF7 are two human hormone-dependent breast cancer cell lines which are widely used as experimental models for in vitro and in vivo (tumor xenografts breast cancer studies. Several proteins involved in cancer development were identified in these cell lines by proteomic analyses. Although these studies reported the proteomic profiles of each cell line, until now, their differential protein expression profiles have not been established. Here, we used two-dimensional gel and mass spectrometry analyses to compare the proteomic profiles of the two cell lines, T47D and MCF7. Our data revealed that more than 164 proteins are differentially expressed between them. According to their biological functions, the results showed that proteins involved in cell growth stimulation, anti-apoptosis mechanisms and cancerogenesis are more strongly expressed in T47D than in MCF7. These proteins include G1/S-specific cyclin-D3 and prohibitin. Proteins implicated in transcription repression and apoptosis regulation, including transcriptional repressor NF-X1, nitrilase homolog 2 and interleukin-10, are, on the contrary, more strongly expressed in MCF7 as compared to T47D. Five proteins that were previously described as breast cancer biomarkers, namely cathepsin D, cathepsin B, protein S100-A14, heat shock protein beta-1 (HSP27 and proliferating cell nuclear antigen (PCNA, are found to be differentially expressed in the two cell lines. A list of differentially expressed proteins between T47D and MCF7 was generated, providing useful information for further studies of breast cancer mechanisms with these cell lines as models.

  11. Proteomic analysis of prolactinoma cells by immuno-laser capture microdissection combined with online two-dimensional nano-scale liquid chromatography/mass spectrometry

    Directory of Open Access Journals (Sweden)

    Chen Luping

    2010-01-01

    Full Text Available Abstract Background Pituitary adenomas, the third most common intracranial tumor, comprise nearly 16.7% of intracranial neoplasm and 25%-44% of pituitary adenomas are prolactinomas. Prolactinoma represents a complex heterogeneous mixture of cells including prolactin (PRL, endothelial cells, fibroblasts, and other stromal cells, making it difficult to dissect the molecular and cellular mechanisms of prolactin cells in pituitary tumorigenesis through high-throughout-omics analysis. Our newly developed immuno-laser capture microdissection (LCM method would permit rapid and reliable procurement of prolactin cells from this heterogeneous tissue. Thus, prolactin cell specific molecular events involved in pituitary tumorigenesis and cell signaling can be approached by proteomic analysis. Results Proteins from immuno-LCM captured prolactin cells were digested; resulting peptides were separated by two dimensional-nanoscale liquid chromatography (2D-nanoLC/MS and characterized by tandem mass spectrometry. All MS/MS spectrums were analyzed by SEQUEST against the human International Protein Index database and a specific prolactinoma proteome consisting of 2243 proteins was identified. This collection of identified proteins by far represents the largest and the most comprehensive database of proteome for prolactinoma. Category analysis of the proteome revealed a widely unbiased access to various proteins with diverse functional characteristics. Conclusions This manuscript described a more comprehensive proteomic profile of prolactinomas compared to other previous published reports. Thanks to the application of immuno-LCM combined with online two-dimensional nano-scale liquid chromatography here permitted identification of more proteins and, to our best knowledge, generated the largest prolactinoma proteome. This enlarged proteome would contribute significantly to further understanding of prolactinoma tumorigenesis which is crucial to the management of

  12. Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

    Science.gov (United States)

    Sacco, Francesca; Silvestri, Alessandra; Posca, Daniela; Pirrò, Stefano; Gherardini, Pier Federico; Castagnoli, Luisa; Mann, Matthias; Cesareni, Gianni

    2016-03-23

    Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin. PMID:27135362

  13. Time-course proteomics dataset monitoring HeLa cells subjected to DTT induced endoplasmic reticulum stress.

    Science.gov (United States)

    Cheng, Zhe; Rendleman, Justin; Vogel, Christine

    2016-09-01

    The data described here provide an analysis of the dynamic response of HeLa cell proteome to dithiothreitol (DTT) inducing stress of the endoplasmic reticulum (ER). During ER stress, accumulation of misfolded and unfolded proteins in the lumen of the ER initiates the Unfolded Protein Response (UPR), resulting in a large-scale redistribution of proteins. We used label-free mass spectrometry to monitor the proteomic changes of HeLa cells during a 30-h time course, monitoring eight time points (0, 0.5, 1, 2, 8, 16, 24, and 30 h). The data are associated with the research article "Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress" [1], which discusses a core dataset of 1237 proteins. Here, we present the extended dataset of 2131 proteins. The raw mass spectrometry data and the analysis results have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PRIDE: PXD002039. PMID:27547793

  14. Data from a comparative proteomic analysis of tumor-derived lung-cancer CD105(+) endothelial cells.

    Science.gov (United States)

    Jin, Hongwei; Cheng, Xiao; Pei, Yihua; Fu, Jianguo; Lyu, Zhi; Peng, Huifang; Yao, Qin; Jiang, Yu; Luo, Lianzhong; Zhuo, Huiqin

    2016-06-01

    Increasing evidence indicates that tumor-derived endothelial cells (TECs) are more relevant for the study of tumor angiogenesis and for screening antiangiogenic drugs than normal ECs (NECs). In this data article, high-purity (>98%) primary CD105(+) NECs and TECs purified from a mouse Lewis lung carcinoma model bearing 0.5 cm tumors were identified using 2D-PAGE and Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All the identified proteins were categorized functionally by Gene Ontology (GO) analysis, and gene-pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, protein-protein interaction networks were also built. The proteomics and bioinformatics data presented here provide novel insights into the molecular characteristics and the early modulation of the TEC proteome in the tumor microenvironment. PMID:27081670

  15. Effects of stem cell therapy on protein profile of parkinsonian rats using an(18) O-labeling quantitative proteomic approach.

    Science.gov (United States)

    Liu, Yahui; Liu, Kefu; Qin, Wei; Liu, Chenghao; Zheng, Xiaowei; Deng, Yulin; Qing, Hong

    2016-03-01

    The application of neural stem cell (NSC) research to neurodegenerative diseases has led to promising clinical trials. Currently, NSC therapy is most promising for Parkinson's disease (PD). We conducted behavioral tests and immunoassays for the profiling of a PD model in rats to assess the therapeutic effects of NSC treatments. Further, using a multiple sample comparison workflow, combined with (18) O-labeled proteome mixtures, we compared the differentially expressed proteins from control, PD, and NSC-treated PD rats. The results were analyzed bioinformatically and verified by Western blot. Based on our initial findings, we believe that the proteomic approach is a valuable tool in evaluating the therapeutic effects of NSC transplantation on neurodegenerative disorders. PMID:26791447

  16. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

    KAUST Repository

    Chan, Yuk-kit

    2015-04-01

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

  17. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

    Directory of Open Access Journals (Sweden)

    Nisha Jain Garg

    2016-02-01

    Full Text Available Trypanosoma cruzi (Tc infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs of normal healthy controls (N/H, n = 30 and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25 or clinically symptomatic (C/S, n = 28 with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol and resolved by two-dimensional gel electrophoresis (2D-GE. After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.

  18. Plasma proteome analysis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma

    Indian Academy of Sciences (India)

    Mee Lee Looi; Saiful Anuar Karsani; Mariati Abdul Rahman; Ahmad Zailani Hatta Mohd Dali; Siti Aishah Md Ali; Wan Zurinah Wan Ngah; Yasmin Anum Mohd Yusof

    2009-12-01

    Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.

  19. Proteomic Analyses of the Effects of Drugs of Abuse on Monocyte-Derived Mature Dendritic Cells

    OpenAIRE

    Jessica L. Reynolds; Supriya D Mahajan; Aalinkeel, Ravikunar; Nair, B.; Sykes, Donald E; Schwartz, Stanley A.

    2009-01-01

    Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that a...

  20. Sperm cell biology: current perspectives and future prospects

    Institute of Scientific and Technical Information of China (English)

    R John Aitken; Ralf R Henkel

    2011-01-01

    @@ Major advances in biomolecular techniques as well as in the sensitivity and accuracy of mass spectrometers are transform-ing the scientific landscape by fueling unprecedented advances in ana-lytical biochemistry-the 'omics revolution, which refers to the study of genes (genomics), transcripts (transcriptomics), proteins (proteo-mics) and the various metabolites (metabolomics). It is now possible to secure inventories of lipids, proteins, metabolites and RNA species in purified cell populations and to determine how these entities change in relation to cellular function.

  1. Combining proteomics and transcriptome sequencing to identify active plant-cell-wall-degrading enzymes in a leaf beetle

    Directory of Open Access Journals (Sweden)

    Kirsch Roy

    2012-11-01

    Full Text Available Abstract Background The primary plant cell wall is a complex mixture of polysaccharides and proteins encasing living plant cells. Among these polysaccharides, cellulose is the most abundant and useful biopolymer present on earth. These polysaccharides also represent a rich source of energy for organisms which have evolved the ability to degrade them. A growing body of evidence suggests that phytophagous beetles, mainly species from the superfamilies Chrysomeloidea and Curculionoidea, possess endogenous genes encoding complex and diverse families of so-called plant cell wall degrading enzymes (PCWDEs. The presence of these genes in phytophagous beetles may have been a key element in their success as herbivores. Here, we combined a proteomics approach and transcriptome sequencing to identify PCWDEs present in larval gut contents of the mustard leaf beetle, Phaedon cochleariae. Results Using a two-dimensional proteomics approach, we recovered 11 protein bands, isolated using activity assays targeting cellulose-, pectin- and xylan-degrading enzymes. After mass spectrometry analyses, a total of 13 proteins putatively responsible for degrading plant cell wall polysaccharides were identified; these proteins belong to three glycoside hydrolase (GH families: GH11 (xylanases, GH28 (polygalacturonases or pectinases, and GH45 (β-1,4-glucanases or cellulases. Additionally, highly stable and proteolysis-resistant host plant-derived proteins from various pathogenesis-related protein (PRs families as well as polygalacturonase-inhibiting proteins (PGIPs were also identified from the gut contents proteome. In parallel, transcriptome sequencing revealed the presence of at least 19 putative PCWDE transcripts encoded by the P. cochleariae genome. All of these were specifically expressed in the insect gut rather than the rest of the body, and in adults as well as larvae. The discrepancy observed in the number of putative PCWDEs between transcriptome and proteome

  2. Quantitative Membrane Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteogenic Differentiation

    DEFF Research Database (Denmark)

    Christiansen, Helle

    . Mesenchymal stem cells are generally isolated based on physical-chemical characteristics such as adherence to plastic, isolating the monocyte fraction. The resultant cultures are often heterogeneous and can contain other cell types, providing a currently poorly defined basis for future clinical use. The...... associated or GPI anchored proteins. A considerable number of CD antigens and GPI anchored proteins were identified and quantified, illustrating a high degree of sub-fractional proteome resolution. We identified and quantified known markers of hMSC (e.g. CD71, CD166, CD105, CD44, CD29, CD90 and CD63) as well...

  3. The Redox Proteome*

    OpenAIRE

    Go, Young-Mi; Jones, Dean P.

    2013-01-01

    The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation,...

  4. Proteomic signatures of human oral epithelial cells in HIV-infected subjects.

    Directory of Open Access Journals (Sweden)

    Elizabeth Yohannes

    Full Text Available The oral epithelium, the most abundant structural tissue lining the oral mucosa, is an important line of defense against infectious microorganisms. HIV infected subjects on highly active antiretroviral therapy (HAART are susceptible to comorbid viral, bacterial and fungal infections in the oral cavity. To provide an assessment of the molecular alterations of oral epithelia potentially associated with susceptibility to comorbid infections in such subjects, we performed various proteomic studies on over twenty HIV infected and healthy subjects. In a discovery phase two Dimensional Difference Gel Electrophoresis (2-D DIGE analyses of human oral gingival epithelial cell (HOEC lysates were carried out; this identified 61 differentially expressed proteins between HIV-infected on HAART subjects and healthy controls. Down regulated proteins in HIV-infected subjects include proteins associated with maintenance of protein folding and pro- and anti-inflammatory responses (e.g., heat-shock proteins, Cryab, Calr, IL-1RA, and Galectin-3-binding protein as well as proteins involved in redox homeostasis and detoxification (e.g., Gstp1, Prdx1, and Ero1. Up regulated proteins include: protein disulfide isomerases, proteins whose expression is negatively regulated by Hsp90 (e.g., Ndrg1, and proteins that maintain cellular integrity (e.g., Vimentin. In a verification phase, proteins identified in the protein profiling experiments and those inferred from Ingenuity Pathway Analysis were analyzed using Western blotting analysis on separate HOEC lysate samples, confirming many of the discovery findings. Additionally in HIV-infected patient samples Heat Shock Factor 1 is down regulated, which explains the reduced heat shock responses, while activation of the MAPK signal transduction cascade is observed. Overall, HAART therapy provides an incomplete immune recovery of the oral epithelial cells of the oral cavity for HIV-infected subjects, and the toxic side effects of

  5. Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-B1

    Energy Technology Data Exchange (ETDEWEB)

    Kurpinski, Kyle; Chu, Julia; Wang, Daojing; Li, Song

    2009-10-12

    Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor {beta}1 (TGF-{beta}1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-{beta}1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-{beta}1-induced protein ig-h3 (BGH3) protein levels by TGF-{beta}1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-{beta}1, but calponin 3 was not significantly regulated by mechanical strain or TGF-{beta}1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-{beta}1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-{beta}1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation.

  6. Are clear cell carcinomas of the ovary and endometrium phenotypically identical? A proteomic analysis.

    Science.gov (United States)

    Fata, Cynthia R; Seeley, Erin H; Desouki, Mohamed M; Du, Liping; Gwin, Katja; Hanley, Krisztina Z; Hecht, Jonathan L; Jarboe, Elke A; Liang, Sharon X; Parkash, Vinita; Quick, Charles M; Zheng, Wenxin; Shyr, Yu; Caprioli, Richard M; Fadare, Oluwole

    2015-10-01

    Phenotypic differences between otherwise similar tumors arising from different gynecologic locations may be highly significant in understanding the underlying driver molecular events at each site and may potentially offer insights into differential responses to treatment. In this study, the authors sought to identify and quantify phenotypic differences between ovarian clear cell carcinoma (OCCC) and endometrial clear cell carcinoma (ECCC) using a proteomic approach. Tissue microarrays were constructed from tumor samples of 108 patients (54 ECCCs and 54 OCCCs). Formalin-fixed samples on microarray slides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry, and 730 spectral peaks were generated from the combined data set. A linear mixed-effect model with random intercept was used to generate 93 (12.7%) peaks that were significantly different between OCCCs and ECCCs at the fold cutoffs of 1.5 and 0.667 and an adjusted P value cutoff of 1.0 × 10(-10). Liquid chromatography-tandem mass spectrometry was performed on selected cores from each group, and peptides identified therefrom were compared with lists of statistically significant peaks from the aforementioned linear mixed-effects model to find matches within 0.2 Da. A total of 53 candidate proteins were thus identified as being differentially expressed in OCCCs and ECCCs, 45 (85%) of which were expressed at higher levels in ECCCs than OCCCs. These proteins were functionally diverse and did not highlight a clearly dominant cellular theme or molecular pathway. Although ECCCs and OCCCs are very similar, some phenotypic differences are demonstrable. Additional studies of these differentially expressed proteins may ultimately clarify the significance of these differences. PMID:26243671

  7. Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure

    Energy Technology Data Exchange (ETDEWEB)

    Roland, Kathleen, E-mail: kathleen.roland@fundp.ac.be [Research Unit in Environmental and Evolutionary Biology (URBE), Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium); Kestemont, Patrick; Hénuset, Laurence; Pierrard, Marie-Aline [Research Unit in Environmental and Evolutionary Biology (URBE), Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium); Raes, Martine; Dieu, Marc [Research Unit in Cellular Biology (URBC) Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium); Silvestre, Frédéric [Research Unit in Environmental and Evolutionary Biology (URBE), Narilis (Namur Research Institute for Lifesciences), University of Namur - FUNDP, Rue de Bruxelles 61, B-5000, Namur (Belgium)

    2013-03-15

    Highlights: ► We have evaluating the toxicity of eel peripheral blood mononuclear cells exposed during 48 h to 10 μg and 1 mg perfluoroctane sulfonate/L. ► After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed. ► 48 different proteins were identified and classified into main functional classes which provide clues on the cellular pathways mainly affected by PFOS. -- Abstract: Since the 1980s, the stocks of European eel have been declining in most of their geographical distribution area. Many factors can be attributed to this decline such as pollution by xenobiotics like perfluorooctane sulfonate (PFOS). This study aimed at evaluating the in vitro toxicity of eel peripheral blood mononuclear cells (PBMC) exposed to PFOS. Exposure time and two concentrations were chosen to avoid cell mortality (48 h exposure at 10 μg PFOS/L and 1 mg PFOS/L). After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed to compare PBMC from the control group with cells exposed to the pollutant. On the 158 spots that were significantly affected by PFOS exposure, a total of 48 different proteins were identified using nano-LCESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. These proteins can be categorized into diverse functional classes, related to cytoskeleton, protein folding, cell signaling, proteolytic pathway and carbohydrate and energy metabolism, which provide clues on the cellular pathways mainly affected by PFOS. Some of the identified proteins are rarely found in other ecotoxicological proteomic studies and could constitute potential biomarkers of exposure to PFOS in fish.

  8. Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Yansheng Liu

    Full Text Available Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA and Multiple reaction monitoring (MRM assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG and Leucine-rich alpha-2-glycoprotein (LRG1, two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

  9. Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

    OpenAIRE

    Maddi, Abhiram; Bowman, Shaun M.; Free, Stephen J.

    2009-01-01

    Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and 6 secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and 9 secreted proteins. Most ...

  10. Proteomic Profiling of Iron Overload-Induced Human Hepatic Cells Reveals Activation of TLR2-Mediated Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Xiang Li

    2016-03-01

    Full Text Available Background: Hepatic iron overload is common in patients who have undergone hematopoietic cell transplantation (HCT and may predispose to peri- and post-HCT toxicity. To better reveal more molecules that might be involved in iron overload-induced liver injury, we utilized proteomics to investigate differentially expressed proteins in iron overload-induced hepatocytes vs. untreated hepatocytes. Methods and Results: HH4 hepatocytes were exposed to ferric ammonium citrate (FAC to establish an in vitro iron overload model. Differentially expressed proteins initiated by the iron overload were studied by two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS analysis. We identified 93 proteins whose quantity statistically significantly changes under excess hepatocyte iron conditions. Gene Ontology (GO analysis showed that these differentially expressed proteins in HH4 cells are involved in various biological process including endocytosis, response to wounding, di-, trivalent inorganic cation homeostasis, inflammatory response, positive regulation of cytokine production, and etc. Meanwhile, proteomics data revealed protein level of TLR2 and IL6ST significantly increased 7 times and 2.9 times, respectively, in iron overloaded HH4 cells. Our subsequent experiments detected that FAC-treated HH4 cells can activate IL6 expression through TLR2-mediated inflammatory responses via the NF-κB pathway. Conclusions: In this study, we demonstrated that iron overload induced hepatocytes triggering TLR2-mediated inflammatory response via NF-κB signaling pathway in HH4 cells.

  11. Comparative proteomic data of M13SV1 human breast epithelial cells and their tumorigenic variants under treatment with estrogenic compounds.

    Science.gov (United States)

    Braeuning, Albert; Schmidt, Claudia; Oberemm, Axel; Lampen, Alfonso

    2016-09-01

    Data from a comparative proteomic analysis of three human breast epithelial cell lines are presented. M13SV1 cells and their tumorigenic derivatives M13SV1-R2-2 and M13SV1-R2-N1 were used. Proteomic data were obtained using 2-dimensional gel electrophoresis and subsequent identification of proteins by MALDI-TOF mass spectrometry. In a second experiment, the three cell lines were treated with different concentrations of the estrogenic compounds β-estradiol or genistein and alterations in protein expression were monitored by 2-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Presented data provide a comprehensive overview of proteomic differences between the three cell lines and their response to estrogenic stimulation. PMID:27331110

  12. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target

    Science.gov (United States)

    Toman, O.; Kabickova, T.; Vit, O.; Fiser, R.; Polakova, K. Machova; Zach, J.; Linhartova, J.; Vyoral, D.; Petrak, J.

    2016-01-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib-resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  13. Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism.

    Science.gov (United States)

    George, Iniga S; Pascovici, Dana; Mirzaei, Mehdi; Haynes, Paul A

    2015-09-01

    Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label-free quantitative shotgun proteomic analysis was performed. A total of 2042 non-redundant proteins were identified from the five temperature points. Fifty-five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold-responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 (http://proteomecentral.proteomexchange.org/dataset/PXD000977). PMID:25959233

  14. Proteomics in Pancreatic Cancer Research

    Science.gov (United States)

    Geng, Ruihui; Li, Zhaoshen; Li, Shude; Gao, Jun

    2011-01-01

    Pancreatic cancer is a highly aggressive malignancy with a poor prognosis and deeply affects the life of people. Therefore, the earlier diagnosis and better treatments are urgently needed. In recent years, the proteomic technologies are well established and growing rapidly and have been widely applied in clinical applications, especially in pancreatic cancer research. In this paper, we attempt to discuss the development of current proteomic technologies and the application of proteomics to the field of pancreatic cancer research. This will explore the potential perspective in revealing pathogenesis, making the diagnosis earlier and treatment. PMID:22084685

  15. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

    Science.gov (United States)

    Garg, Nisha Jain; Soman, Kizhake V; Zago, Maria P; Koo, Sue-Jie; Spratt, Heidi; Stafford, Susan; Blell, Zinzi N; Gupta, Shivali; Nuñez Burgos, Julio; Barrientos, Natalia; Brasier, Allan R; Wiktorowicz, John E

    2016-02-01

    Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, psurvival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy. PMID:26919708

  16. Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure.

    Science.gov (United States)

    Roland, Kathleen; Kestemont, Patrick; Hénuset, Laurence; Pierrard, Marie-Aline; Raes, Martine; Dieu, Marc; Silvestre, Frédéric

    2013-03-15

    Since the 1980s, the stocks of European eel have been declining in most of their geographical distribution area. Many factors can be attributed to this decline such as pollution by xenobiotics like perfluorooctane sulfonate (PFOS). This study aimed at evaluating the in vitro toxicity of eel peripheral blood mononuclear cells (PBMC) exposed to PFOS. Exposure time and two concentrations were chosen to avoid cell mortality (48 h exposure at 10 μg PFOS/L and 1mg PFOS/L). After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed to compare PBMC from the control group with cells exposed to the pollutant. On the 158 spots that were significantly affected by PFOS exposure, a total of 48 different proteins were identified using nano-LCESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. These proteins can be categorized into diverse functional classes, related to cytoskeleton, protein folding, cell signaling, proteolytic pathway and carbohydrate and energy metabolism, which provide clues on the cellular pathways mainly affected by PFOS. Some of the identified proteins are rarely found in other ecotoxicological proteomic studies and could constitute potential biomarkers of exposure to PFOS in fish. PMID:23261670

  17. Atypical carcinoid and large cell neuroendocrine carcinoma of the lung: a proteomic dataset from formalin-fixed archival samples.

    Science.gov (United States)

    Tanca, Alessandro; Addis, Maria Filippa; Pisanu, Salvatore; Abbondio, Marcello; Pagnozzi, Daniela; Eccher, Albino; Rindi, Guido; Cossu-Rocca, Paolo; Uzzau, Sergio; Fanciulli, Giuseppe

    2016-06-01

    Here we present a dataset generated using formalin-fixed paraffin-embedded archival samples from two rare lung neuroendocrine tumor subtypes (namely, two atypical carcinoids, ACs, and two large-cell neuroendocrine carcinomas, LCNECs). Samples were subjected to a shotgun proteomics pipeline, comprising full-length protein extraction, SDS removal through spin columns, in solution trypsin digestion, long gradient liquid chromatography peptide separation and LTQ-Orbitrap mass spectrometry analysis. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR http://www.peptideatlas.org/PASS/PASS00375. PMID:27054153

  18. Proteomic and meatbolomic analysis of smooth muscle cells derived from the arterial media and adventitial progenitors of apolipoprotein E-deficient mice

    NARCIS (Netherlands)

    Mayr, M; Zampetaki, A.; Sidibe, A.; Mayr, U.; Yin, X.; Souza, A.I. de; Chung, Y.L.; Madhu, B.; Quax, P.H.; Hu, Y.; Griffiths, J.R.; Xu, Q.

    2008-01-01

    We have recently demonstrated that stem cell antigen 1-positive (Sca-1(+)) progenitors exist in the vascular adventitia of apolipoprotein E-deficient (apoE(-/-)) mice and contribute to smooth muscle cell (SMC) accumulation in vein graft atherosclerosis. Using a combined proteomic and metabolomic app

  19. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics

    DEFF Research Database (Denmark)

    Ong, S.E.; Blagoev, B.; Kratchmarova, I.;

    2002-01-01

    . Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non......Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures......-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred...

  20. Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies

    Czech Academy of Sciences Publication Activity Database

    Žižková, Martina; Suchá, Rita; Tylečková, Jiřina; Jarkovská, Karla; Mairychová, Kateřina; Kotrčová, Eva; Marsala, M.; Gadher, S. J.; Kovářová, Hana

    2015-01-01

    Roč. 12, č. 1 (2015), s. 83-95. ISSN 1478-9450 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell therapy * immunomodulation * neural stem cell differentiation * neural subpopulation * neurodegenerative disease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.896, year: 2014

  1. Transplantation proteomics

    OpenAIRE

    Traum, Avram Z.; Schachter, Asher D.

    2005-01-01

    The field of proteomics is developing at a rapid pace in the post-genome era. Translational proteomics investigations aim to apply a combination of established methods and new technologies to learn about protein expression profiles predictive of clinical events, therapeutic response, and underlying mechanisms. However, in contrast to genetic studies and in parallel with gene expression studies, the dynamic nature of the proteome in conjunction with the challenges of accounting for post-transl...

  2. Quantitative proteomics analysis reveals glutamine deprivation activates fatty acid β-oxidation pathway in HepG2 cells.

    Science.gov (United States)

    Long, Baisheng; Muhamad, Rodiallah; Yan, Guokai; Yu, Jie; Fan, Qiwen; Wang, Zhichang; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

    2016-05-01

    Glutamine, a multifunctional amino acid, functions in nutrient metabolism, energy balance, apoptosis, and cell proliferation. Lipid is an important nutrient and controls a broad range of physiological processes. Previous studies have demonstrated that glutamine can affect lipolysis and lipogenesis, but the effect of glutamine on the detailed lipid metabolism remains incompletely understood. Here, we applied the quantitative proteomics approach to estimate the relative abundance of proteins in HepG2 cells treated by glutamine deprivation. The results showed that there were 212 differentially abundant proteins in response to glutamine deprivation, including 150 significantly increased proteins and 62 significantly decreased proteins. Interestingly, functional classification showed that 43 differentially abundant proteins were related to lipid metabolism. Further bioinformatics analysis and western blotting validation revealed that lipid accumulation may be affected by β-oxidation of fatty acid induced by glutamine deprivation in HepG2 cells. Together, our results may provide the potential for regulating lipid metabolism by glutamine in animal production and human nutrition. The MS data have been deposited to the ProteomeXchange Consortium with identifier PXD003387. PMID:26837383

  3. Apoptosis of Candida albicans during the Interaction with Murine Macrophages: Proteomics and Cell-Death Marker Monitoring.

    Science.gov (United States)

    Cabezón, Virginia; Vialás, Vital; Gil-Bona, Ana; Reales-Calderón, Jose A; Martínez-Gomariz, Montserrat; Gutiérrez-Blázquez, Dolores; Monteoliva, Lucía; Molero, Gloria; Ramsdale, Mark; Gil, Concha

    2016-05-01

    Macrophages may induce fungal apoptosis to fight against C. albicans, as previously hypothesized by our group. To confirm this hypothesis, we analyzed proteins from C. albicans cells after 3 h of interaction with macrophages using two quantitative proteomic approaches. A total of 51 and 97 proteins were identified as differentially expressed by DIGE and iTRAQ, respectively. The proteins identified and quantified were different, with only seven in common, but classified in the same functional categories. The analyses of their functions indicated that an increase in the metabolism of amino acids and purine nucleotides were taking place, while the glycolysis and translation levels dropped after 3 h of interaction. Also, the response to oxidative stress and protein translation were reduced. In addition, seven substrates of metacaspase (Mca1) were identified (Cdc48, Fba1, Gpm1, Pmm1, Rct1, Ssb1, and Tal1) as decreased in abundance, plus 12 proteins previously described as related to apoptosis. Besides, the monitoring of apoptotic markers along 24 h of interaction (caspase-like activity, TUNEL assay, and the measurement of ROS and cell examination by transmission electron microscopy) revealed that apoptotic processes took place for 30% of the fungal cells, thus supporting the proteomic results and the hypothesis of macrophages killing C. albicans by apoptosis. PMID:27048922

  4. Protein translation machinery holds a key for transition of planktonic cells to biofilm state in Enterococcus faecalis: A proteomic approach.

    Science.gov (United States)

    Qayyum, Shariq; Sharma, Divakar; Bisht, Deepa; Khan, Asad U

    2016-06-10

    Enterococcus faecalis is a member of human gut microflora causing nosocomial infection involving biofilm formation. Ethyl methyl sulfonate induced mutants were analysed using crystal violet assay, SEM and CLSM microscopy which confirmed AK-E12 as biofilm efficient and AK-F6 as biofilm deficient mutants. Growth curve pattern revealed AK-E12 was fast growing whereas, AK-F6 was found slow growing mutant. 2D-Electrophorosis and MALDI-TOF analysis revealed over and underexpression of many translation-elongation associated proteins in mutants compared to wild type. Protein translation elongation factor G, translation elongation factor Tu and ribosomal subunit interface proteins were underexpressed and UTP-glucose-1-phosphate uridylyl transferase and cell division protein divIVA were overexpressed in AK-E12 as compared to wild type. In AK-F6, except 10 kDa chaperonin which was over-expressed other selected proteins were found to be suppressed. RT-PCR confirmed proteomic data except for the translation elongation factor G which showed contradictory data of proteome expression in AK-E12. Protein-protein interaction networks were constructed using STRING 10.0 which demonstrated strong connection of translation-elongation proteins with other proteins. Hence, it concludes from the data that translation elongation factors are important in transition of planktonic cells to biofilm cells in Enterococcus faecalis. PMID:27144316

  5. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  6. Stem cells: a plant biology perspective

    NARCIS (Netherlands)

    Scheres, B.J.G.

    2005-01-01

    A recent meeting at the Juan March Foundation in Madrid, Spain brought together plant biologists to discuss the characteristics of plant stem cells that are unique and those that are shared by stem cells from the animal kingdom

  7. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.

    Science.gov (United States)

    Zielke, Ryszard A; Wierzbicki, Igor H; Weber, Jacob V; Gafken, Philip R; Sikora, Aleksandra E

    2014-05-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  8. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha;

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  9. Integrated Proteomic and Glycoproteomic Analyses of Prostate Cancer Cells Reveal Glycoprotein Alteration in Protein Abundance and Glycosylation.

    Science.gov (United States)

    Shah, Punit; Wang, Xiangchun; Yang, Weiming; Toghi Eshghi, Shadi; Sun, Shisheng; Hoti, Naseruddin; Chen, Lijun; Yang, Shuang; Pasay, Jered; Rubin, Abby; Zhang, Hui

    2015-10-01

    Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in

  10. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen; Mann, Matthias; Jensen, Ole Nørregaard; Kassem, Moustapha

    2005-01-01

    of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and......One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely...... in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral...

  11. Mapping the chromatographic behavior of a cell proteome utilizing orthogonal routines: the influence of feedstock pH

    Directory of Open Access Journals (Sweden)

    Rosa Cabrera

    2008-08-01

    Full Text Available Surface charge, molecular weight, and folding state are known to influence protein chromatographic behavior onto ion-exchangers. Experimentally, information related to such factors can be gathered via two-dimensional electrophoretic (2-DE methods. The separation behavior depicted by the insect cultured-cells proteome, which is an important host for recombinant protein production, was explored in this study. Experimental evidence showed a correlation between apparent isoelectric point distributions and the mobile phase conductivity. It was observed that the information contained in the isoelectric point (pI value(s obtained with a 2-DE routine showed a good correlation with the IEX chromatographic behavior, for a number of commercial adsorbents. This correlation was observed irrespective of the pH of the feedstock within the range 6 to 8. An initial prediction of protein ion-exchange chromatographic behavior could be possible utilizing an experimental approach based on the mentioned orthogonal methods. This technique is providing information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.Keywords: Insect cells, proteome, chromatography, ion-exchange, bioprocessingReceived: 3 August 2008 / Received in revised form: 15 August 2008, Accepted: 19 August 2008, Published online: 20 August 2008

  12. Plant Cell Wall Proteomics: Mass Spectrometry Data, a Trove for Research on Protein Structure/Function Relationships

    Institute of Scientific and Technical Information of China (English)

    Cécile Albenne; Hervé Canut; Georges Boudart; Yu Zhang; Héléne San Clemente; Rafael Pont-Lezica; Elisabeth Jamet

    2009-01-01

    Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organ-elles. In particular, mass spectrometry (MS) analysis of gel-separated proteins produces data not only for protein identi-fication, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics of Arabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins (H/PRPs) is arduous, different types of MS spectra were exploited to identify and characterize an H/PRR Matu-ration events in a couple of cell wall proteins (CWPs) were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.

  13. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

  14. Comparative proteomic analysis of CD34(+) cells in bone marrow between severe aplastic anemia and normal control.

    Science.gov (United States)

    Qi, Weiwei; Fu, Rong; Wang, Huaquan; Liu, Chunyan; Ren, Yue; Shao, Yuanyuan; Shao, Zonghong

    2016-01-01

    Severe aplastic anemia (SAA) is an autoimmune disease with destruction of hematopoietic cells by activated T lymphocytes. However, the precise mechanism of cytotoxicity T cells recognizing and attacking CD34(+) cells remains unclear. Here, we investigated the proteome of CD34(+) cells in SAA patients to further explore the pathogenesis of SAA. CD34(+) cells from 29 SAA patients and 20 health controls were isolated by magnetic activated cell sorting. The protein of CD34(+) cells were examined by iTRAQ labeling combination of multidimensional liquid chromatography and tandem mass spectrometry. A total of 156 differential expression proteins in CD34(+) cells were identified. Compared with health controls, 53 proteins were up-regulated and 103 proteins were down-regulated in SAA patients. Specifically, abnormal expression of proteasome subunits, histone variants, dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit (DAD1) and ATPase inhibitor, mitochondrial isoform 1 precursor(IF1) may relate to the hyperfunction of immune responses and excessive apoptosis of SAA CD34(+) cells. PMID:27086042

  15. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    Science.gov (United States)

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

  16. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  17. A novel method for sample preparation of fresh lung cancer tissue for proteomics analysis by tumor cell enrichment and removal of blood contaminants

    Directory of Open Access Journals (Sweden)

    Orre Lotta

    2010-02-01

    Full Text Available Abstract Background In-depth proteomics analyses of tumors are frequently biased by the presence of blood components and stromal contamination, which leads to large experimental variation and decreases the proteome coverage. We have established a reproducible method to prepare freshly collected lung tumors for proteomics analysis, aiming at tumor cell enrichment and reduction of plasma protein contamination. We obtained enriched tumor-cell suspensions (ETS from six lung cancer cases (two adenocarcinomas, two squamous-cell carcinomas, two large-cell carcinomas and from two normal lung samples. The cell content of resulting ETS was evaluated with immunocytological stainings and compared with the histologic pattern of the original specimens. By means of a quantitative mass spectrometry-based method we evaluated the reproducibility of the sample preparation protocol and we assessed the proteome coverage by comparing lysates from ETS samples with the direct lysate of corresponding fresh-frozen samples. Results Cytological analyses on cytospin specimens showed that the percentage of tumoral cells in the ETS samples ranged from 20% to 70%. In the normal lung samples the percentage of epithelial cells was less then 10%. The reproducibility of the sample preparation protocol was very good, with coefficient of variation at the peptide level and at the protein level of 13% and 7%, respectively. Proteomics analysis led to the identification of a significantly higher number of proteins in the ETS samples than in the FF samples (244 vs 109, respectively. Albumin and hemoglobin were among the top 5 most abundant proteins identified in the FF samples, showing a high contamination with blood and plasma proteins, whereas ubiquitin and the mitochondrial ATP synthase 5A1 where among the top 5 most abundant proteins in the ETS samples. Conclusion The method is feasible and reproducible. We could obtain a fair enrichment of cells but the major benefit of the method

  18. Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

    Directory of Open Access Journals (Sweden)

    Reshu Saxena

    Full Text Available Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1 through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01 in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1, VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61 and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein

  19. Global MS-Based Proteomics Drug Profiling.

    Science.gov (United States)

    Carvalho, Ana Sofia; Matthiesen, Rune

    2016-01-01

    DNA-based technologies such as RNAi, chemical-genetic profiling, or gene expression profiling by DNA microarrays combined with other biochemical methods are established strategies for surveying drug mechanisms. Such approaches can provide mechanistic information on how drugs act and affect cellular pathways. By studying how cancer cells compensate for the drug treatment, novel targets used in a combined treatment can be designed. Furthermore, toxicity effects on cells not targeted can be obtained on a molecular level. For example, drug companies are particularly interested in studying the molecular side effects of drugs in the liver. In addition, experiments with the purpose of elucidating liver toxicity can be studied using samples obtained from animal models exposed to different concentrations of a drug over time. More recently considerable advances in mass spectrometry (MS) technologies and bioinformatics tools allows informative global drug profiling experiments to be performed at a cost comparable to other large-scale technologies such as DNA-based technologies. Moreover, MS-based proteomics provides an additional layer of information on the dynamic regulation of proteins translation and particularly protein degradation. MS-based proteomics approaches combined with other biochemical methods delivers information on regulatory networks, signaling cascades, and metabolic pathways upon drug treatment. Furthermore, MS-based proteomics can provide additional information on single amino acid polymorphisms, protein isoform distribution, posttranslational modifications, and subcellular localization. In this chapter, we will share our experience using MS based proteomics as a pharmacoproteomics strategy to characterize drug mechanisms of action in single drug therapy or in multidrug combination. Finally, the emergence of integrated proteogenomics analysis, such as "The Cancer Genome Atlas" program, opened interesting perspectives to extend this approach to drug target

  20. Clinical perspectives of cancer stem cell research in radiation oncology

    International Nuclear Information System (INIS)

    Radiotherapy has a proven potential to eradicate cancer stem cells which is reflected by its curative potential in many cancer types. Considerable progress has been made in identification and biological characterisation of cancer stem cells during the past years. Recent biological findings indicate significant inter- and intratumoural and functional heterogeneity of cancer stem cells and lead to more complex models which have potential implications for radiobiology and radiotherapy. Clinical evidence is emerging that biomarkers of cancer stem cells may be prognostic for the outcome of radiotherapy in some tumour entities. Perspectives of cancer stem cell based research for radiotherapy reviewed here include their radioresistance compared to the mass of non-cancer stem cells which form the bulk of all tumour cells, implications for image- and non-image based predictive bio-assays of the outcome of radiotherapy and a combination of novel systemic treatments with radiotherapy

  1. DOE perspective on fuel cells in transportation

    Energy Technology Data Exchange (ETDEWEB)

    Kost, R.

    1996-04-01

    Fuel cells are one of the most promising technologies for meeting the rapidly growing demand for transportation services while minimizing adverse energy and environmental impacts. This paper reviews the benefits of introducing fuel cells into the transportation sector; in addition to dramatically reduced vehicle emissions, fuel cells offer the flexibility than use petroleum-based or alternative fuels, have significantly greater energy efficiency than internal combustion engines, and greatly reduce noise levels during operation. The rationale leading to the emphasis on proton-exchange-membrane fuel cells for transportation applications is reviewed as are the development issues requiring resolution to achieve adequate performance, packaging, and cost for use in automobiles. Technical targets for power density, specific power, platinum loading on the electrodes, cost, and other factors that become increasingly more demanding over time have been established. Fuel choice issues and pathways to reduced costs and to a renewable energy future are explored. One such path initially introduces fuel cell vehicles using reformed gasoline while-on-board hydrogen storage technology is developed to the point of allowing adequate range (350 miles) and refueling convenience. This scenario also allows time for renewable hydrogen production technologies and the required supply infrastructure to develop. Finally, the DOE Fuel Cells in Transportation program is described. The program, whose goal is to establish the technology for fuel cell vehicles as rapidly as possible, is being implemented by means of the United States Fuel Cell Alliance, a Government-industry alliance that includes Detroit`s Big Three automakers, fuel cell and other component suppliers, the national laboratories, and universities.

  2. Proteomics analysis of human oligodendroglioma proteome.

    Science.gov (United States)

    Khaghani-Razi-Abad, Solmaz; Hashemi, Mehrdad; Pooladi, Mehdi; Entezari, Maliheh; Kazemi, Elham

    2015-09-10

    Proteomics analyses enable the identification and quantitation of proteins. From a purely clinical perspective, the application of proteomics based on innovations, may greatly affect the future management of malignant brain tumors. This optimism is based on four main reasons: diagnosis, prognosis, selection of targeted therapy based on molecular profile of the brain tumor and monitoring therapeutic response, or resistance. We extracted the proteins of tumor and normal brain tissues, and then evaluated the protein purity by Bradford test. In this study, we separated the proteins by two-dimensional (2DG) gel electrophoresis methods. Then spots were analyzed, compared using statistical data and specific software and were identified by pH isoelectric, molecular weights and data banks. The protein profiles were determined using 2D gel electrophoresis and MALDI TOF/TOF mass spectrometry approaches. Simple statistical tests were used to establish a putative hierarchy in which the change in protein level was ranked according to a cut-off point with pProteomics is a powerful way to identifying multiple proteins which are altered following a neuropharmacological intervention in a CNS disease. PMID:26002447

  3. Artificial Dendritic Cells: Multi-faceted Perspectives

    CERN Document Server

    Greensmith, Julie

    2009-01-01

    Dendritic cells are the crime scene investigators of the human immune system. Their function is to correlate potentially anomalous invading entities with observed damage to the body. The detection of such invaders by dendritic cells results in the activation of the adaptive immune system, eventually leading to the removal of the invader from the host body. This mechanism has provided inspiration for the development of a novel bio-inspired algorithm, the Dendritic Cell Algorithm. This algorithm processes information at multiple levels of resolution, resulting in the creation of information granules of variable structure. In this chapter we examine the multi-faceted nature of immunology and how research in this field has shaped the function of the resulting Dendritic Cell Algorithm. A brief overview of the algorithm is given in combination with the details of the processes used for its development. The chapter is concluded with a discussion of the parallels between our understanding of the human immune system a...

  4. Limbal stem cell transplantation: current perspectives

    Directory of Open Access Journals (Sweden)

    Atallah MR

    2016-04-01

    Full Text Available Marwan Raymond Atallah, Sotiria Palioura, Victor L Perez, Guillermo Amescua Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA Abstract: Regeneration of the corneal surface after an epithelial insult involves division, migration, and maturation of a specialized group of stem cells located in the limbus. Several insults, both intrinsic and extrinsic, can precipitate destruction of the delicate microenvironment of these cells, resulting in limbal stem cell deficiency (LSCD. In such cases, reepithelialization fails and conjunctival epithelium extends across the limbus, leading to vascularization, persistent epithelial defects, and chronic inflammation. In partial LSCD, conjunctival epitheliectomy, coupled with amniotic membrane transplantation, could be sufficient to restore a healthy surface. In more severe cases and in total LSCD, stem cell transplantation is currently the best curative option. Before any attempts are considered to perform a limbal stem cell transplantation procedure, the ocular surface must be optimized by controlling causative factors and comorbid conditions. These factors include adequate eyelid function or exposure, control of the ocular surface inflammatory status, and a well-lubricated ocular surface. In cases of unilateral LSCD, stem cells can be obtained from the contralateral eye. Newer techniques aim at expanding cells in vitro or in vivo in order to decrease the need for large limbal resection that may jeopardize the “healthy” eye. Patients with bilateral disease can be treated using allogeneic tissue in combination with systemic immunosuppressive therapy. Another emerging option for this subset of patients is the use of noncorneal cells such as mucosal grafts. Finally, the use of keratoprosthesis is reserved for patients who are not candidates for any of the aforementioned options, wherein the choice of the type of keratoprosthesis depends on

  5. Single cell genomics: advances and future perspectives.

    OpenAIRE

    Macaulay, Iain C.; Thierry Voet

    2014-01-01

    Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organis...

  6. Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis

    Institute of Scientific and Technical Information of China (English)

    Xing'an Li; Yinjiu Zhang; Yihong Hu; Ming Chang; Tao Liu; Danping Wang; Yu Zhang; Lei Zhang; Linsen Hu

    2008-01-01

    Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled.Protcomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 μM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and caldum-binding protein I were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.

  7. Mesenchymal stem cells in diabetes treatment: progress and perspectives

    Directory of Open Access Journals (Sweden)

    Yu CHENG

    2016-08-01

    Full Text Available Diabetes is a chronic metabolic disorder caused by relative or absolute insulin deficient or reduced sensitivity of target cells to insulin. Mesenchymal stem cells (MSCs are adult stem cells with multiple differentiation potential, self-renewable and immunoregulatory properties. Accumulating evidences from clinic or animal experiments recent years showed that MSCs infusion could ameliorate hyperglycemia in diabetes. The research progress of MSCs in diabetes treatment is summarized and a corresponding perspective is herewith proposed in present paper. DOI: 10.11855/j.issn.0577-7402.2016.07.16

  8. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target.

    Science.gov (United States)

    Toman, O; Kabickova, T; Vit, O; Fiser, R; Polakova, K Machova; Zach, J; Linhartova, J; Vyoral, D; Petrak, J

    2016-09-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib‑resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  9. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

    Directory of Open Access Journals (Sweden)

    Rust Steven

    2013-02-01

    Full Text Available Abstract Background The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. Methods The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. Results All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated

  10. Increased protein synthesis by cells exposed to a 1,800-MHz radio-frequency mobile phone electromagnetic field, detected by proteome profiling

    OpenAIRE

    Gerner, Christopher; Haudek, Verena; Schandl, Ulla; Bayer, Editha; Gundacker, Nina; Hutter, Hans Peter; Mosgoeller, Wilhelm

    2010-01-01

    Purpose To investigate whether or not low intensity radio frequency electromagnetic field exposure (RF-EME) associated with mobile phone use can affect human cells, we used a sensitive proteome analysis method to study changes in protein synthesis in cultured human cells. Methods Four different cell kinds were exposed to 2 W/kg specific absorption rate in medium containing 35S-methionine/cysteine, and autoradiography of 2D gel spots was used to measure the increased synthesis of individual pr...

  11. Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

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    Schriemer David C

    2010-11-01

    Full Text Available Abstract Background Papaver somniferum (opium poppy is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. Results A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. Conclusions The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a

  12. Effect of adipose-derived stromal cells and BMP12 on intrasynovial tendon repair: A biomechanical, biochemical, and proteomics study.

    Science.gov (United States)

    Gelberman, Richard H; Shen, Hua; Kormpakis, Ioannis; Rothrauff, Benjamin; Yang, Guang; Tuan, Rocky S; Xia, Younan; Sakiyama-Elbert, Shelly; Silva, Matthew J; Thomopoulos, Stavros

    2016-04-01

    The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. PMID:26445383

  13. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    Directory of Open Access Journals (Sweden)

    José B Gama

    2014-08-01

    Full Text Available Buruli ulcer (BU is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1 and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1. In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis.

  14. Proteomic analysis of the action of the Mycobacterium ulcerans toxin mycolactone: targeting host cells cytoskeleton and collagen.

    Science.gov (United States)

    Gama, José B; Ohlmeier, Steffen; Martins, Teresa G; Fraga, Alexandra G; Sampaio-Marques, Belém; Carvalho, Maria A; Proença, Fernanda; Silva, Manuel T; Pedrosa, Jorge; Ludovico, Paula

    2014-08-01

    Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans. The tissue damage characteristic of BU lesions is known to be driven by the secretion of the potent lipidic exotoxin mycolactone. However, the molecular action of mycolactone on host cell biology mediating cytopathogenesis is not fully understood. Here we applied two-dimensional electrophoresis (2-DE) to identify the mechanisms of mycolactone's cellular action in the L929 mouse fibroblast proteome. This revealed 20 changed spots corresponding to 18 proteins which were clustered mainly into cytoskeleton-related proteins (Dync1i2, Cfl1, Crmp2, Actg1, Stmn1) and collagen biosynthesis enzymes (Plod1, Plod3, P4ha1). In line with cytoskeleton conformational disarrangements that are observed by immunofluorescence, we found several regulators and constituents of both actin- and tubulin-cytoskeleton affected upon exposure to the toxin, providing a novel molecular basis for the effect of mycolactone. Consistent with these cytoskeleton-related alterations, accumulation of autophagosomes as well as an increased protein ubiquitination were observed in mycolactone-treated cells. In vivo analyses in a BU mouse model revealed mycolactone-dependent structural changes in collagen upon infection with M. ulcerans, associated with the reduction of dermal collagen content, which is in line with our proteomic finding of mycolactone-induced down-regulation of several collagen biosynthesis enzymes. Our results unveil the mechanisms of mycolactone-induced molecular cytopathogenesis on exposed host cells, with the toxin compromising cell structure and homeostasis by inducing cytoskeleton alterations, as well as disrupting tissue structure, by impairing the extracellular matrix biosynthesis. PMID:25101965

  15. Quantitative proteomics and transcriptomics reveals metabolic differences in attracting and non-attracting human-in-mouse glioma stem cell xenografts and stromal cells

    Directory of Open Access Journals (Sweden)

    Norelle C. Wildburger

    2015-09-01

    Full Text Available Bone marrow-derived human mesenchymal stem cells (BM-hMSCs show promise as cell-based delivery vehicles for anti-glioma therapeutics, due to innate tropism for gliomas. However, in clinically relevant human-in-mouse glioma stem cell xenograft models, BM-hMSCs tropism is variable. We compared the proteomic profile of cancer and stromal cells in GSCXs that attract BM-hMSCs (“attractors” with those to do not (“non-attractors” to identify pathways that may modulate BM-hMSC homing, followed by targeted transcriptomics. The results provide the first link between fatty acid metabolism, glucose metabolism, ROS, and N-glycosylation patterns in attractors. Reciprocal expression of these pathways in the stromal cells suggests microenvironmental cross-talk.

  16. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    International Nuclear Information System (INIS)

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: ► Melanogenesis stimulation by L-tyrosine+NH4Cl in B16-F10 melanoma cells increases ROS levels. ► Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. ► Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. ► RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  17. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: grmartinez@ufpr.br [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)

    2012-09-10

    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  18. Proteomics - new analytical approaches

    International Nuclear Information System (INIS)

    Full text: Recent developments in the sequencing of the human genome have indicated that the number of coding gene sequences may be as few as 30,000. It is clear, however, that the complexity of the human species is dependent on the much greater diversity of the corresponding protein complement. Estimates of the diversity (discrete protein species) of the human proteome range from 200,000 to 300,000 at the lower end to 2,000,000 to 3,000,000 at the high end. In addition, proteomics (the study of the protein complement to the genome) has been subdivided into two main approaches. Global proteomics refers to a high throughput examination of the full protein set present in a cell under a given environmental condition. Focused proteomics refers to a more detailed study of a restricted set of proteins that are related to a specified biochemical pathway or subcellular structure. While many of the advances in proteomics will be based on the sequencing of the human genome, de novo characterization of protein microheterogeneity (glycosylation, phosphorylation and sulfation as well as the incorporation of lipid components) will be required in disease studies. To characterize these modifications it is necessary to digest the protein mixture with an enzyme to produce the corresponding mixture of peptides. In a process analogous to sequencing of the genome, shot-gun sequencing of the proteome is based on the characterization of the key fragments produced by such a digest. Thus, a glycopeptide and hence a specific glycosylation motif will be identified by a unique mass and then a diagnostic MS/MS spectrum. Mass spectrometry will be the preferred detector in these applications because of the unparalleled information content provided by one or more dimensions of mass measurement. In addition, highly efficient separation processes are an absolute requirement for advanced proteomic studies. For example, a combination of the orthogonal approaches, HPLC and HPCE, can be very powerful

  19. Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue-Derived Stem Cells, and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis.

    Science.gov (United States)

    Pires, Ana O; Mendes-Pinheiro, Barbara; Teixeira, Fábio G; Anjo, Sandra I; Ribeiro-Samy, Silvina; Gomes, Eduardo D; Serra, Sofia C; Silva, Nuno A; Manadas, Bruno; Sousa, Nuno; Salgado, Antonio J

    2016-07-15

    The use of human mesenchymal stem cells (hMSCs) has emerged as a possible therapeutic strategy for CNS-related conditions. Research in the last decade strongly suggests that MSC-mediated benefits are closely related with their secretome. Studies published in recent years have shown that the secretome of hMSCs isolated from different tissue sources may present significant variation. With this in mind, the present work performed a comparative proteomic-based analysis through mass spectrometry on the secretome of hMSCs derived from bone marrow (BMSCs), adipose tissue (ASCs), and human umbilical cord perivascular cells (HUCPVCs). The results revealed that BMSCs, ASCs, and HUCPVCs differed in their secretion of neurotrophic, neurogenic, axon guidance, axon growth, and neurodifferentiative proteins, as well as proteins with neuroprotective actions against oxidative stress, apoptosis, and excitotoxicity, which have been shown to be involved in several CNS disorder/injury processes. Although important changes were observed within the secretome of the cell populations that were analyzed, all cell populations shared the capability of secreting important neuroregulatory molecules. The difference in their secretion pattern may indicate that their secretome is specific to a condition of the CNS. Nevertheless, the confirmation that the secretome of MSCs isolated from different tissue sources is rich in neuroregulatory molecules represents an important asset not only for the development of future neuroregenerative strategies but also for their use as a therapeutic option for human clinical trials. PMID:27226274

  20. New generation solar cells: Concepts, trends and perspectives

    OpenAIRE

    Ragoussi, Maria Eleni; Torres, Tomás

    2015-01-01

    Organic, dye-sensitized and perovskite solar cell technologies have triggered widespread interest in recent years due to their very promising potential towards a high solar electricity future. A number of important milestones have marked the roadmap of each sector on the way to today's outstanding performances, but there still remains plenty of scope for further improvement. The most influential landmarks, together with basic concepts and future perspectives, are unraveled in this review

  1. Induced pluripotent stem cells:origins, applications, and future perspectives

    Institute of Scientific and Technical Information of China (English)

    Jing ZHAO; Wen-jie JIANG; Chen SUN; Cong-zhe HOU; Xiao-mei YANG; Jian-gang GAO

    2013-01-01

    Embryonic stem (ES) cells are widely used for different purposes, including gene targeting, celltherapy, tissue repair, organ regeneration, and so on. However, studies and applications of ES cells are hindered by ethical issues regarding cellsources. To circumvent ethical disputes, great efforts have been taken to generate ES cel-like cells, which are not derived from the inner cellmass of blastocyst-stage embryos. In 2006, Yamanaka et al. first re-programmed mouse embryonic fibroblasts into ES cell-like cells cal ed induced pluripotent stem (iPS) cells. About one year later, Yamanaka et al. and Thomson et al. independently reprogrammed human somatic cells into iPS cells. Since the first generation of iPS cells, they have now been derived from quite a few different kinds of celltypes. In particular, the use of peripheral blood facilitates research on iPS cells because of safety, easy availability, and plenty of cellsources. Now iPS cells have been used for celltherapy, disease modeling, and drug discovery. In this review, we describe the generations, applications, potential issues, and future perspectives of iPS cells.

  2. A Proteomics Analysis to Evaluate Cytotoxicity in NRK-52E Cells Caused by Unmodified Nano-Fe3O4

    Directory of Open Access Journals (Sweden)

    Yi-Reng Lin

    2014-01-01

    Full Text Available We synthesized unmodified Fe3O4 nanoparticles (NPs with particles size from 10 nm to 100 nm. We cultured NRK-52E cell lines (rat, kidney and treated with Fe3O4 NPs to investigate and evaluate the cytotoxicity of NPs for NRK-52E cells. Through global proteomics analysis using dimethyl labeling techniques and liquid phase chromatography coupled with a tandem mass spectrometer (LC-MS/MS, we characterized 435 proteins including the programmed cell death related proteins, ras-related proteins, glutathione related proteins, and the chaperone proteins such as heat shock proteins, serpin H1, protein disulfide-isomerase A4, endoplasmin, and endoplasmic reticulum resident proteins. From the statistical data of identified proteins, we believed that NPs treatment causes cell death and promotes expression of ras-related proteins. In order to avoid apoptosis, NRK-52E cell lines induce a series of protective effects such as glutathione related proteins to reduce reactive oxygen species (ROS, and chaperone proteins to recycle damaged proteins. We suggested that, in the indigenous cellular environment, Fe3O4 NPs treatment induced an antagonistic effect for cell lines to go to which avoids apoptosis.

  3. Computing the functional proteome

    DEFF Research Database (Denmark)

    O'Brien, Edward J.; Palsson, Bernhard

    2015-01-01

    -Models). Recent expansions in network content to encompass proteome synthesis have resulted in models of metabolism and protein expression (ME-Models). ME-Models advance the predictions possible with constraint-based models from network flux states to the spatially resolved molecular composition of a cell....... Specifically, ME-Models enable the prediction of transcriptome and proteome allocation and limitations, and basal expression states and regulatory needs. Continued expansion in reconstruction content and constraints will result in an increasingly refined representation of cellular composition and behavior....

  4. Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

    Directory of Open Access Journals (Sweden)

    Klopfleisch Robert

    2012-06-01

    Full Text Available Abstract Background Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition. Results Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Conclusions Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect.

  5. Systems Biology Perspectives on Minimal and Simpler Cells

    Science.gov (United States)

    Xavier, Joana C.; Patil, Kiran Raosaheb

    2014-01-01

    SUMMARY The concept of the minimal cell has fascinated scientists for a long time, from both fundamental and applied points of view. This broad concept encompasses extreme reductions of genomes, the last universal common ancestor (LUCA), the creation of semiartificial cells, and the design of protocells and chassis cells. Here we review these different areas of research and identify common and complementary aspects of each one. We focus on systems biology, a discipline that is greatly facilitating the classical top-down and bottom-up approaches toward minimal cells. In addition, we also review the so-called middle-out approach and its contributions to the field with mathematical and computational models. Owing to the advances in genomics technologies, much of the work in this area has been centered on minimal genomes, or rather minimal gene sets, required to sustain life. Nevertheless, a fundamental expansion has been taking place in the last few years wherein the minimal gene set is viewed as a backbone of a more complex system. Complementing genomics, progress is being made in understanding the system-wide properties at the levels of the transcriptome, proteome, and metabolome. Network modeling approaches are enabling the integration of these different omics data sets toward an understanding of the complex molecular pathways connecting genotype to phenotype. We review key concepts central to the mapping and modeling of this complexity, which is at the heart of research on minimal cells. Finally, we discuss the distinction between minimizing the number of cellular components and minimizing cellular complexity, toward an improved understanding and utilization of minimal and simpler cells. PMID:25184563

  6. Identifying cytotoxic T cell epitopes from genomic and proteomic information: "The human MHC project."

    DEFF Research Database (Denmark)

    Lauemøller, S L; Kesmir, C; Corbet, S L;

    2000-01-01

    processing, as these become available. The ability to translate the accumulating primary sequence databases in terms of immune recognition should enable scientists and clinicians to analyze any protein of interest for the presence of potentially immunogenic epitopes. The computational tools to scan entire...... discrimination, even at the peptide level. It is not surprising that peptides are key targets of the immune system. It follows that proteomes can be translated into immunogens once it is known how the immune system generates and handles peptides. Recent advances have identified many of the basic principles...

  7. Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans.

    Science.gov (United States)

    Maddi, Abhiram; Bowman, Shaun M; Free, Stephen J

    2009-10-01

    Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS-PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall. PMID:19555771

  8. Corneal stem cells and tissue engineering: Current advancesand future perspectives

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Major advances are currently being made in regenerativemedicine for cornea. Stem cell-based therapiesrepresent a novel strategy that may substituteconventional corneal transplantation, albeit there aremany challenges ahead given the singularities of eachcellular layer of the cornea. This review recapitulatesthe current data on corneal epithelial stem cells,corneal stromal stem cells and corneal endothelialcell progenitors. Corneal limbal autografts containingepithelial stem cells have been transplanted in humansfor more than 20 years with great successful rates,and researchers now focus on ex vivo cultures andother cell lineages to transplant to the ocular surface.A small population of cells in the corneal endotheliumwas recently reported to have self-renewal capacity,although they do not proliferate in vivo . Two mainobstacles have hindered endothelial cell transplantationto date culture protocols and cell delivery methods tothe posterior cornea in vivo . Human corneal stromalstem cells have been identified shortly after therecognition of precursors of endothelial cells. Stromalstem cells may have the potential to provide a directcell-based therapeutic approach when injected tocorneal scars. Furthermore, they exhibit the ability todeposit organized connective tissue in vitro and maybe useful in corneal stroma engineering in the future.Recent advances and future perspectives in the field arediscussed.

  9. Proteome-wide analysis of SUMO2 targets in response to pathological DNA replication stress in human cells

    DEFF Research Database (Denmark)

    Bursomanno, Sara; Beli, Petra; Khan, Asif M;

    2015-01-01

    subfamily. SUMO2/3, in contrast to SUMO1, are predominantly involved in the cellular response to certain stresses, including heat shock. Substantial evidence from studies in yeast has shown that SUMOylation plays an important role in the regulation of DNA replication and repair. Here, we report a proteomic...... analysis of proteins modified by SUMO2 in response to DNA replication stress in S phase in human cells. We have identified a panel of 22 SUMO2 targets with increased SUMOylation during DNA replication stress, many of which play key functions within the DNA replication machinery and/or in the cellular...... response to DNA damage. Interestingly, POLD3 was found modified most significantly in response to a low dose aphidicolin treatment protocol that promotes common fragile site (CFS) breakage. POLD3 is the human ortholog of POL32 in budding yeast, and has been shown to act during break-induced recombinational...

  10. Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann;

    2014-01-01

    Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line...... T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated...... quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ- proteomics. We identified several proteins linked...

  11. Characterization of T-cell responses to conserved regions of the HIV-1 proteome in BALB/c mice.

    Science.gov (United States)

    Ondondo, Beatrice; Abdul-Jawad, Sultan; Bridgeman, Anne; Hanke, Tomáš

    2014-11-01

    A likely requirement for a protective vaccine against human immunodeficiency virus type 1 (HIV-1)/AIDS is, in addition to eliciting antibody responses, induction of effective T cells. To tackle HIV-1 diversity by T-cell vaccines, we designed an immunogen, HIVconsv, derived from the most functionally conserved regions of the HIV-1 proteome and demonstrated its high immunogenicity in humans and rhesus macaques when delivered by regimens combining plasmid DNA, nonreplicating simian (chimpanzee) adenovirus ChAdV-63, and nonreplicating modified vaccinia virus Ankara (MVA) as vectors. Here, we aimed to increase the decision power for iterative improvements of this vaccine strategy in the BALB/c mouse model. First, we found that prolonging the period after the ChAdV63.HIVconsv prime up to 6 weeks increased the frequencies of HIV-1-specific, gamma interferon (IFN-γ)-producing T cells induced by the MVA.HIVconsv boost. Induction of strong responses allowed us to map comprehensively the H-2(d)-restricted T-cell responses to these regions and identified 8 HIVconsv peptides, of which three did not contain a previously described epitope and were therefore considered novel. Induced effector T cells were oligofunctional and lysed sensitized targets in vitro. Our study therefore provides additional tools for studying and optimizing vaccine regimens in this commonly used small animal model, which will in turn guide vaccine improvements in more expensive nonhuman primate and human clinical trials. PMID:25230940

  12. Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

    Directory of Open Access Journals (Sweden)

    Paul Majkut

    Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

  13. Perspective in infertility: the ovarian stem cells.

    Science.gov (United States)

    Silvestris, Erica; D'Oronzo, Stella; Cafforio, Paola; D'Amato, Giuseppe; Loverro, Giuseppe

    2015-01-01

    Infertility is a medical and social condition that affects millions of women worldwide and is today considered so far as a new disease. A considerable progress has been recently pursued in the field of the reproductive medicine and the infertility treatment may account for novel and modern procedures such as in vitro oocyte fertilization, egg donation, pregnancy surrogacy and preimplantation diagnosis. However, great interest has lately been reserved to the ovarian stem cells (OSCs) whose existence in woman ovaries has been proven. OSCs are thus suitable for developmental studies in infertility and in other clinical applications as endocrine derangements due to premature ovarian failure, or for infertility treatment after cancer chemotherapies, as well as in restoring the hormonal balance in postmenopausal age. PMID:26250560

  14. An historical perspective on cell mechanics.

    Science.gov (United States)

    Pelling, Andrew E; Horton, Michael A

    2008-04-01

    The physical properties of the protoplasm have long been of interest, and today, several intricate methods, including atomic force microscopy, have been employed in studies of cellular mechanics. However, many current concepts and experimental approaches actually have their beginnings over 300 years ago. Unfortunately, these pioneering studies have been all but forgotten. In this paper, we have reviewed some of the early literature on cellular mechanics to place modern work within an historical framework. It is clear that with current nanoscience approaches, modern experiments employing cell indentation, manipulation, particle rheology and micro- or nano-needle poking are now quantifying mechanical properties which were only qualitatively described 100 years ago. Aside from the variety of approaches our predecessors have employed to understand cellular mechanics, we feel an understanding of the past will help to propel nanoscience into the future. As nanophysiology and nanomedicine are developing, we as a community should take time to consider the early roots of these fields. PMID:18064487

  15. Accessing to the minor proteome of red blood cells through the influence of the nanoparticle surface properties on the corona composition

    Directory of Open Access Journals (Sweden)

    Zaccaria A

    2015-03-01

    Full Text Available Affif Zaccaria,1,* Florence Roux-Dalvai,2,3,* Ali Bouamrani,1 Adrien Mombrun,1 Pascal Mossuz,4 Bernard Monsarrat,2,3 François Berger1 1Clinatec CEA-LETI, Grenoble, 2CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale, 3Université de Toulouse, UPS, IPBS, Toulouse, 4TIMC-Therex UMR 5525 CNRS, UJF, CHU Grenoble, Grenoble, France *These authors contributed equally to this work Abstract: Nanoparticle (NP–protein interactions in complex samples have not yet been clearly understood. Nevertheless, several studies demonstrated that NP’s physicochemical features significantly impact on the protein corona composition. Taking advantage of the NP potential to harvest different subsets of proteins, we assessed for the first time the capacity of three kinds of superparamagnetic NPs to highlight the erythrocyte minor proteome. Using both qualitative and quantitative proteomics approaches, nano-liquid chromatography–tandem mass spectrometry allowed the identification of 893 different proteins, confirming the reproducible capacity of NPs to increase the number of identified proteins, through a reduction of the sample concentration range and the capture of specific proteins on the three different surfaces. These NP-specific protein signatures revealed significant differences in their isoelectric point and molecular weight. Moreover, this NP strategy offered a deeper access to the erythrocyte proteome highlighting several signaling pathways implicated in important erythrocyte functions. The automated potentiality, the reproducibility, and the low-consuming sample demonstrate the strong compatibility of our strategy for large-scale clinical studies and may become a standardized sample preparation in future erythrocyte-associated proteomics studies. Keywords: nanoparticles, red blood cells, mass spectrometry, quantitative proteomics, protein corona, minor proteome 

  16. Proteome-wide effect of 17-β-Estradiol and Lipoxin A4 in an endometriotic epithelial cell line

    Directory of Open Access Journals (Sweden)

    Jonathan eSobel

    2016-01-01

    Full Text Available Endometriosis affects approximately 10% of the women of reproductive age. This chronic gynecological inflammatory disease results in a decreased quality of life for patients, with the main symptoms including chronic pelvic pain and infertility. The steroid hormone 17-β Estradiol (E2 plays a key role in the pathology. Our previous studies showed that the anti-inflammatory lipid Lipoxin A4 (LXA4 acts an estrogen receptor alpha agonist in endometrial epithelial cells, inhibiting certain E2-mediated effects. LXA4 also prevents the progression of endometriosis in a mouse model via anti-proliferative mechanisms and by impacting mediators downstream of ER signaling. The aim of the present study was therefore to examine global proteomic changes evoked by E2 and LXA4 in endometriotic epithelial cells. E2 impacted a greater number of proteins in endometriotic epithelial cells than LXA4. Interestingly, the combination of E2 and LXA4 resulted in a reduced number of regulated proteins, with LXA4 mediating a suppressive effect on E2-mediated signaling. These proteins are involved in diverse pathways of relevance to endometriosis pathology and metabolism, including mRNA translation, growth, proliferation, proteolysis and immune responses. In summary, this study sheds light on novel pathways involved in endometriosis pathology and furthers understanding of signaling pathways activated by estrogenic molecules in endometriotic epithelial cells.

  17. Cell Wall Biology: Perspectives from Cell Wall Imaging

    Institute of Scientific and Technical Information of China (English)

    Kieran J.D.Lee; Susan E.Marcus; J.Paul Knox

    2011-01-01

    Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth,are major repositories for photosynthetically accumulated carbon,and,in addition,impact greatly on the human use of plants. Land plant cell walls contain in the region of a dozen major polysaccharide structures that are mostly encompassed by cellulose,hemicelluloses,and pectic polysaccharides. During the evolution of land plants,polysaccharide diversification appears to have largely involved structural elaboration and diversification within these polysaccharide groups. Cell wall chemistry is well advanced and a current phase of cell wall science is aimed at placing the complex polysaccharide chemistry in cellular contexts and developing a detailed understanding of cell wall biology. Imaging cell wall glycomes is a challenging area but recent developments in the establishment of cell wall molecular probe panels and their use in high throughput procedures are leading to rapid advances in the molecular understanding of the spatial heterogeneity of individual cell walls and also cell wall differences at taxonomic levels. The challenge now is to integrate this knowledge of cell wall heterogeneity with an understanding of the molecular and physiological mechanisms that underpin cell wall properties and functions.

  18. Comparative proteomics analysis of apoptotic Spodoptera frugiperda cells during p35 knockout Autographa californica multiple nucleopolyhedrovirus infection.

    Science.gov (United States)

    Yu, Qian; Xiong, Youhua; Liu, Jianliang; Wang, Qin; Qiu, Yuanxin; Wen, Dongling

    2016-06-01

    Infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutants lacking a functional p35 gene can induce host cell apoptosis, which provides the possibility to use the potential of these viruses in the biological control of pest insects. Nonetheless, the proteomics or the protein changes of Spodoptera frugiperda (Sf9) cells infected with p35 knockout AcMNPV have not yet been studied. To further improve the use of AcMNPV, we set out to analyze the protein composition and protein changes of Sf9 cells of different infection stages by isobaric tag for relative and absolute quantification (iTRAQ) techniques. A total of 4004 sf9 proteins were identified by iTRAQ. After comparation of the significantly expressed 483 proteins from p35koAcMNPV-infected Sf9 cells and the significantly expressed 413 proteins from wtAcMNPV-infected Sf9 cells, we found that 226 proteins were specific to p35koAcMNPV-infected Sf9 cells. The 226 proteins were categorized according to GO classification for insects and were categorized into: biological processes, molecular functions and cellular components. Of interest, the most up-regulated proteins related to Epstein-Barr virus infection, RNA transport, Calcium signaling pathway, cGMP-PKG signaling pathway, oxidative phosphorylation and N-Glycan biosynthesis. Determination of the protein changes in p35 knockout AcMNPV-infected Sf9 cells would facilitate the better use of this virus-host cell interaction in pest insect control and other related fields. PMID:26922645

  19. Quantitative proteomic analysis of the inhibitory effects of CIL-102 on viability and invasiveness in human glioma cells

    International Nuclear Information System (INIS)

    CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone), the major active agent of the alkaloid derivative, has been demonstrated to exert anticancer effects. Herein, we present an investigation focused on the identification of the target(s) of CIL-102's action and the mechanism of its action in apoptotic and anti-invasive pathways. Proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to assess changes in the expression of relevant protein treatment with CIL-102 that resulted in the inhibition of viability and invasion. Our results demonstrate that CIL-102 treatment of U87 cells decreased cell proliferation and invasiveness. CIL-102 dose-dependent induction of apoptosis and inhibitory invasiveness were accompanied by sustained phosphorylation of JNK1/2 and p70S6K as well as generation of the reactive oxygen species. In addition, differential proteins displayed between CIL-102-treated and untreated U87 were determined and validated. There were 11 differentially expressed proteins between the CIL-102-treated and untreated groups. Furthermore, we demonstrated that CIL-102 inhibited cancer cell proliferation and reduced anti-invasion properties by up-regulating the levels of FUMH (Fumarate hydratase). The investigation demonstrated that there was an increase in the cellular levels of FUMH in the CIL-102 reduction in viability and invasion via the activation of JNK1/2 and mTOR signaling modules. NAC administration and shRNA FUMH conferred resistance to CIL-102-inhibited HIF1α and MMP-2 levels via inhibition of JNK1/2 and mTOR activation. We concluded that CIL-102-induced an apoptosis cascade and decreased aggressiveness in astrocytoma cells by modulation of mitochondria function, providing a new mechanism for CIL-102 treatment. - Highlights: • We found the effect of CIL-102 on neuroblastoma cells. • Fumarate hydratase as a CIL-102's target by proteomic differential displays. • CIL

  20. Quantitative proteomic analysis of the inhibitory effects of CIL-102 on viability and invasiveness in human glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Teng, Chih-Chuan [Institute of Nursing and Department of Nursing, Chang Gung University of Science and Technology, Taiwan (China); Chronic Diseases and Health Promotion Research Center, CGUST, Taiwan (China); Institute of Basic Medicine Science, National Cheng Kung University, Tainan, Taiwan (China); Kuo, Hsing-Chun [Institute of Nursing and Department of Nursing, Chang Gung University of Science and Technology, Taiwan (China); Chronic Diseases and Health Promotion Research Center, CGUST, Taiwan (China); Department of Medical Research China Medical University Hospital, Taichung, Taiwan (China); Sze, Chun-I, E-mail: szec@mail.ncku.edu.tw [Institute of Basic Medicine Science, Department of Cell Biology and Anatomy and Pathology, National Cheng Kung University, Tainan, Taiwan (China)

    2013-11-01

    CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone), the major active agent of the alkaloid derivative, has been demonstrated to exert anticancer effects. Herein, we present an investigation focused on the identification of the target(s) of CIL-102's action and the mechanism of its action in apoptotic and anti-invasive pathways. Proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to assess changes in the expression of relevant protein treatment with CIL-102 that resulted in the inhibition of viability and invasion. Our results demonstrate that CIL-102 treatment of U87 cells decreased cell proliferation and invasiveness. CIL-102 dose-dependent induction of apoptosis and inhibitory invasiveness were accompanied by sustained phosphorylation of JNK1/2 and p70S6K as well as generation of the reactive oxygen species. In addition, differential proteins displayed between CIL-102-treated and untreated U87 were determined and validated. There were 11 differentially expressed proteins between the CIL-102-treated and untreated groups. Furthermore, we demonstrated that CIL-102 inhibited cancer cell proliferation and reduced anti-invasion properties by up-regulating the levels of FUMH (Fumarate hydratase). The investigation demonstrated that there was an increase in the cellular levels of FUMH in the CIL-102 reduction in viability and invasion via the activation of JNK1/2 and mTOR signaling modules. NAC administration and shRNA FUMH conferred resistance to CIL-102-inhibited HIF1α and MMP-2 levels via inhibition of JNK1/2 and mTOR activation. We concluded that CIL-102-induced an apoptosis cascade and decreased aggressiveness in astrocytoma cells by modulation of mitochondria function, providing a new mechanism for CIL-102 treatment. - Highlights: • We found the effect of CIL-102 on neuroblastoma cells. • Fumarate hydratase as a CIL-102's target by proteomic differential

  1. Transcriptional, proteomic, and metabolic responses to lithium in galactose-grown yeast cells

    DEFF Research Database (Denmark)

    Bro, Christoffer; Regenberg, Birgitte; Lagniel, G.; Labarre, J.; Montero-Lomeli, M.; Nielsen, Jens

    2003-01-01

    Lithium is highly toxic to yeast when grown in galactose medium mainly because phosphoglucomutase, a key enzyme of galactose metabolism, is inhibited. We studied the global protein and gene expression profiles of Saccharomyces cerevisiae grown in galactose in different time intervals after addition...... of lithium. These results were related to physiological studies where both secreted and intracellular metabolites were determined. Microarray analysis showed that 664 open reading frames were down-regulated and 725 up-regulated in response to addition of lithium. Genes involved in transcription......, translation, and nucleotide metabolism were down-regulated at the transcriptional level, whereas genes responsive to different stresses as well as genes from energy reserve metabolism and monosaccharide metabolism were up-regulated. Compared with the proteomic data, 26% of the down-regulated and 48% of the up...

  2. Reassessing ecdysteroidogenic cells from the cell membrane receptors’ perspective

    OpenAIRE

    Alexandros Alexandratos; Panagiotis Moulos; Ioannis Nellas; Konstantinos Mavridis; Dedos, Skarlatos G.

    2016-01-01

    Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We ...

  3. At a glance: Proteomics in China

    Institute of Scientific and Technical Information of China (English)

    HE FuChu

    2011-01-01

    Proteomics is a new science that focuses on the comprehensive analysis of proteins in intact organisms or in molecule machineries,organelles,cells,tissues,or organs.It has become an important area of interests in life sciences and has propelled the rapid development of cutting-edge biotechnology in the 21st century.In response to this,the Human Proteome Organization (HUPO) was launched in 2001.The mission of HUPO is to advocate and promote proteomics worldwide and to initiate the Human Proteome Project (HPP) to decode the human genome and to establish the proteomic basis of human physiology and pathology.Eleven projects including the Human Liver Proteome Project (HLPP) led by China are under way.Governments,multinational companies,particularly pharmaceutical and analytical instrument companies,as well as the genomic company Celera Genomics,have invested heavily,hoping to seize the huge potential of proteomics.=He Fuchu,PhD,is a Member of the Chinese Academy of Sciences,a Member of the Academy of Sciences for the Developing World,and is currently the Director of the State Key Laboratory of Proteomics.He is the President of the Beijing Proteome Research Center and a Professor at the Beijing Institute of Radiation Medicine.He Fuchu is a council member of the Human Proteome Organization (HUPO),co-chair (inaugural chair) of the HUPO Human Liver Proteome Project (HLPP),the vice-president of AOHUPO,and the president of CNHUPO.He received his B.S.degree in genetics from Fudan University,Shanghai,in 1982 and earned his M.S.degree in biochemistry and his PhD in cell biology from the Beijing Institute of Radiation Medicine.His major fields of research are proteomics,genomics,bioinformatics and systems biology,with a special interest in liver physiology and pathology.He is a senior editor of Proteomics and Proteomics-Clinical Application and is an editorial board member of Molecular & Cellular Proteomics and the Journal of Proteome Research and an executive editor of the

  4. Reassessing ecdysteroidogenic cells from the cell membrane receptors' perspective.

    Science.gov (United States)

    Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G

    2016-01-01

    Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects. PMID:26847502

  5. Reassessing ecdysteroidogenic cells from the cell membrane receptors’ perspective

    Science.gov (United States)

    Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G.

    2016-01-01

    Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects. PMID:26847502

  6. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    Science.gov (United States)

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-01-01

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  7. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

    Directory of Open Access Journals (Sweden)

    Fei Ye

    Full Text Available Chronic hepatitis C virus (HCV infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1, Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1, carboxylesterase 1 (CES1, vimentin, Proteasome activator complex subunit1 (PSME1, and Cathepsin B (CTSB were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

  8. Correlation between Phylogroups and Intracellular Proteomes of Propionibacterium acnes and Differences in the Protein Expression Profiles between Anaerobically and Aerobically Grown Cells

    Directory of Open Access Journals (Sweden)

    Itaru Dekio

    2013-01-01

    Full Text Available Propionibacterium acnes is one of the dominant commensals on the human skin and also an opportunistic pathogen in relation to acne, sarcoidosis, prostate cancer, and various infections. Recent investigations using housekeeping and virulence genes have revealed that the species consists of three major evolutionary clades (types I, II, and III. In order to investigate protein expression differences between these phylogroups, proteomic profiles of 21 strains of P. acnes were investigated. The proteins extracted from cells cultured under anaerobic and aerobic conditions were analysed using a SELDI-TOF mass spectrometer, high-resolution capillary gel electrophoresis, and LC-MS/ MS. The SELDI spectral profiles were visualised as a heat map and a dendrogram, which resulted in four proteomic groups. Strains belonging to type I were represented in the proteome Group A, while Group B contained type III strains. Groups C and D contained mixtures of types I and II. Each of these groups was not influenced by differences in culture conditions. Under anoxic growth conditions, a type IB strain yielded high expressions of some proteins, such as methylmalonyl-CoA epimerase and the Christie-Atkins-Munch-Petersen (CAMP factor. The present study revealed good congruence between genomic and proteomic data suggesting that the microenvironment of each subtype may influence protein expression.

  9. Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

    DEFF Research Database (Denmark)

    Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N; Ditzel, Henrik J

    2009-01-01

    proteins that could be quantified from 40 to 93%. Repeated LC-MS/MS analyses (3-4 times) of each sample increased the number of identified proteins by 60%. The use of Percoll/sucrose density separation allowed subfractionation of membranes leading to enrichment of membrane proteins (66%) and reduction from...... 33% to only 16% of protein from other membranous organelles such as endoplasmatic reticulum, Golgi, and mitochondria. In total, our optimized methods resulted in 1919 protein identifications (corresponding to 826 at similarity level 80% (SL80); 1145 (509 at SL80) were identified by two or more...... peptides of which 622 (300 at SL80) were membrane proteins. The quantitative proteomic analysis identified 16 cell surface proteins as potential markers of the ability of breast cancer cells to form distant metastases....

  10. Proteome-Wide Quantitation by SILAC

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2010-01-01

    Ongoing improvements in instrumentation, fractionation techniques, and enrichment procedures have dramatically increased the coverage of the proteome achievable via LC-MS/MS-based methodologies, opening the call for approaches to quantitatively assess differences at a proteome-wide scale. Stable...... isotope labeling by amino acids in cell culture (SILAC) has emerged as a powerful and versatile approach for proteome-wide quantitation by mass spectrometry. SILAC utilizes the cells' own metabolism to incorporate isotopically labeled amino acids into its proteome which can be mixed with the proteome of...... detailed procedure for performing SILAC-based experiment for proteome-wide quantitation, including a protocol for optimizing SILAC labeling. We also provide an update on the most recent developments of this technique....

  11. Quantitative Proteomic Analysis of the Human Nucleolus.

    Science.gov (United States)

    Bensaddek, Dalila; Nicolas, Armel; Lamond, Angus I

    2016-01-01

    Recent years have witnessed spectacular progress in the field of mass spectrometry (MS)-based quantitative proteomics, including advances in instrumentation, chromatography, sample preparation methods, and experimental design for multidimensional analyses. It is now possible not only to identify most of the protein components of a cell proteome in a single experiment, but also to describe additional proteome dimensions, such as protein turnover rates, posttranslational modifications, and subcellular localization. Furthermore, by comparing the proteome at different time points, it is possible to create a "time-lapse" view of proteome dynamics. By combining high-throughput quantitative proteomics with detailed subcellular fractionation protocols and data analysis techniques it is also now possible to characterize in detail the proteomes of specific subcellular organelles, providing important insights into cell regulatory mechanisms and physiological responses. In this chapter we present a reliable workflow and protocol for MS-based analysis and quantitation of the proteome of nucleoli isolated from human cells. The protocol presented is based on a SILAC analysis of human MCF10A-Src-ER cells with analysis performed on a Q-Exactive Plus Orbitrap MS instrument (Thermo Fisher Scientific). The subsequent chapter describes how to process the resulting raw MS files from this experiment using MaxQuant software and data analysis procedures to evaluate the nucleolar proteome using customized R scripts. PMID:27576725

  12. Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

    Science.gov (United States)

    Lyupina, Yulia V; Zatsepina, Olga G; Serebryakova, Marina V; Erokhov, Pavel A; Abaturova, Svetlana B; Kravchuk, Oksana I; Orlova, Olga V; Beljelarskaya, Svetlana N; Lavrov, Andrey I; Sokolova, Olga S; Mikhailov, Victor S

    2016-06-01

    Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection. PMID:26945516

  13. Chromosome replication, cell growth, division and shape: a personal perspective.

    Science.gov (United States)

    Zaritsky, Arieh; Woldringh, Conrad L

    2015-01-01

    The origins of Molecular Biology and Bacterial Physiology are reviewed, from our personal standpoints, emphasizing the coupling between bacterial growth, chromosome replication and cell division, dimensions and shape. Current knowledge is discussed with historical perspective, summarizing past and present achievements and enlightening ideas for future studies. An interactive simulation program of the bacterial cell division cycle (BCD), described as "The Central Dogma in Bacteriology," is briefly represented. The coupled process of transcription/translation of genes encoding membrane proteins and insertion into the membrane (so-called transertion) is invoked as the functional relationship between the only two unique macromolecules in the cell, DNA and peptidoglycan embodying the nucleoid and the sacculus respectively. We envision that the total amount of DNA associated with the replication terminus, so called "nucleoid complexity," is directly related to cell size and shape through the transertion process. Accordingly, the primary signal for cell division transmitted by DNA dynamics (replication, transcription and segregation) to the peptidoglycan biosynthetic machinery is of a physico-chemical nature, e.g., stress in the plasma membrane, relieving nucleoid occlusion in the cell's center hence enabling the divisome to assemble and function between segregated daughter nucleoids. PMID:26284044

  14. Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin

    Czech Academy of Sciences Publication Activity Database

    Kubíková, I.; Konečná, H.; Šedo, O.; Zdráhal, Z.; Řehulka, Pavel; Hříbková, H.; Řehulková, Helena; Hampl, Aleš; Chmelík, Josef; Dvořák, Petr

    2009-01-01

    Roč. 11, č. 3 (2009), s. 330-340. ISSN 1465-3249 R&D Projects: GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z40310501; CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : human embryonic stem cell * hESC * proteomic profiling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.204, year: 2009

  15. Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells.

    Science.gov (United States)

    Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

    2016-01-01

    The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells. PMID:27180817

  16. 2D-DIGE proteomic analysis of mesenchymal stem cell cultured on the elasticity-tunable hydrogels.

    Science.gov (United States)

    Kuboki, Thasaneeya; Kantawong, Fahsai; Burchmore, Richard; Dalby, Matthew J; Kidoaki, Satoru

    2012-01-01

    The present study focuses on mechanotransduction in mesenchymal stem cells (MSCs) in response to matrix elasticity. By using photocurable gelatinous gels with tunable stiffness, proteomic profiles of MSCs cultured on tissue culture plastic, soft (3 kPa) and stiff (52 kPa) matrices were deciphered using 2-dimensional differential in-gel analysis (2D-DIGE). The DIGE data, tied to immunofluorescence, indicated abundance and organization changes in the cytoskeletonal proteins as well as differential regulation of important signaling-related proteins, stress-responsing proteins and also proteins involved in collagen synthesis. The major CSK proteins including actin, tubulin and vimentin of the cells cultured on the gels were remarkably changed their expressions. Significant down-regulation of α-tubulin and β-actin can be observed on gel samples in comparison to the rigid tissue culture plates. The expression abundance of vimentin appeared to be highest in the MSCs cultured on hard gels. These results suggested that the substrate stiffness significantly affects expression balances in cytoskeletal proteins of MSCs with some implications to cellular tensegrity. PMID:22971925

  17. Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

    Directory of Open Access Journals (Sweden)

    Silvia Rodríguez-Lombardero

    2014-07-01

    Full Text Available Sky1 is the only member of the SR (Serine–Arginine protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

  18. Non-biased enrichment does not improve quantitative proteomic delineation of reovirus T3D-infected HeLa cell protein alterations

    Directory of Open Access Journals (Sweden)

    Jieyuan eJiang

    2012-09-01

    Full Text Available Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy stable isotopic forms of lysine and arginine (SILAC and infected cells with reovirus strain T3D. Cells were harvested at 24 hours post-infection. Heavy-labeled infected and light-labeled mock-infected cells were mixed together 1:1. Cells were then divided into cytosol and nuclear fractions and each fraction analyzed, both by standard 2D-HPLC/MS, and also after each fraction had been reacted with a random hexapeptide library (Proteominer® beads to attempt to enrich for low-abundance cellular proteins. A total of 2736 proteins were identified by 2 or more peptides at >99% confidence, of which 66 were significantly up-regulated and 67 were significantly down-regulated. Up-regulated proteins included those involved in antimicrobial and antiviral responses, GTPase activity, nucleotide binding, interferon signaling, and enzymes associated with energy generation. Down-regulated proteins included those involved in cell and biological adhesion, regulation of cell proliferation, structural molecule activity, and numerous molecular binding activities. Comparisons of the r2 correlations, degree of dataset overlap, and numbers of peptides detected suggest that non-biased enrichment approaches may not provide additional data to allow deeper quantitative and comparative mining of complex proteomes.

  19. Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium

    Directory of Open Access Journals (Sweden)

    Renes Johan

    2007-04-01

    Full Text Available Abstract Background In vitro models are indispensable study objects in the fields of cell and molecular biology, with advantages such as accessibility, homogeneity of the cell population, reproducibility, and growth rate. The Caco-2 cell line, originating from a colon carcinoma, is a widely used in vitro model for small intestinal epithelium. Cancer cells have an altered metabolism, making it difficult to infer their representativity for the tissue from which they are derived. This study was designed to compare the protein expression pattern of Caco-2 cells with the patterns of intestinal epithelial cells from human small and large intestine. HT-29 intestinal cells, Hep G2 liver cells and TE 671 muscle cells were included too, the latter two as negative controls. Results Two-dimensional gel electrophoresis was performed on each tissue and cell line protein sample. Principal component and cluster analysis revealed that global expression of intestinal epithelial scrapings differed from that of intestinal epithelial cell lines. Since all cultured cell lines clustered together, this finding was ascribed to an adaptation of cells to culture conditions and their tumor origin, and responsible proteins were identified by mass spectrometry. When investigating the profiles of Caco-2 cells and small intestinal cells in detail, a considerable overlap was observed. Conclusion Numerous proteins showed a similar expression in Caco-2 cells, HT-29 cells, and both the intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo. In addition, several biologically significant proteins are expressed at comparable levels in Caco-2 cells and small intestinal scrapings, indicating the usability of this in vitro model. Caco-2 cells, however, appear to over-express as well as under-express certain proteins, which needs to be considered by scientists using this cell line. Hence, care should be taken to prevent misinterpretation of

  20. Evaluation of reverse phase protein array (RPPA)-based pathway-activation profiling in 84 non-small cell lung cancer (NSCLC) cell lines as platform for cancer proteomics and biomarker discovery.

    Science.gov (United States)

    Ummanni, Ramesh; Mannsperger, Heiko A; Sonntag, Johanna; Oswald, Marcus; Sharma, Ashwini K; König, Rainer; Korf, Ulrike

    2014-05-01

    The reverse phase protein array (RPPA) approach was employed for a quantitative analysis of 71 cancer-relevant proteins and phosphoproteins in 84 non-small cell lung cancer (NSCLC) cell lines and by monitoring the activation state of selected receptor tyrosine kinases, PI3K/AKT and MEK/ERK1/2 signaling, cell cycle control, apoptosis, and DNA damage. Additional information on NSCLC cell lines such as that of transcriptomic data, genomic aberrations, and drug sensitivity was analyzed in the context of proteomic data using supervised and non-supervised approaches for data analysis. First, the unsupervised analysis of proteomic data indicated that proteins clustering closely together reflect well-known signaling modules, e.g. PI3K/AKT- and RAS/RAF/ERK-signaling, cell cycle regulation, and apoptosis. However, mutations of EGFR, ERBB2, RAF, RAS, TP53, and PI3K were found dispersed across different signaling pathway clusters. Merely cell lines with an amplification of EGFR and/or ERBB2 clustered closely together on the proteomic, but not on the transcriptomic level. Secondly, supervised data analysis revealed that sensitivity towards anti-EGFR drugs generally correlated better with high level EGFR phosphorylation than with EGFR abundance itself. High level phosphorylation of RB and high abundance of AURKA were identified as candidates that can potentially predict sensitivity towards the aurora kinase inhibitor VX680. Examples shown demonstrate that the RPPA approach presents a useful platform for targeted proteomics with high potential for biomarker discovery. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. PMID:24361481

  1. Proteomic biomarkers of peripheral blood mononuclear cells obtained from postmenopausal women undergoing an intervention with soy isoflavones

    NARCIS (Netherlands)

    Fuchs, D.; Vafeiadou, K.; Hall, W.L.; Daniel, H.; Williams, C.M.; Schroot, J.H.; Wenzel, U.

    2007-01-01

    Background: The incidence of cardiovascular diseases increases after menopause, and soy consumption is suggested to inhibit disease development. Objective: The objective was to identify biomarkers of response to a dietary supplementation with an isoflavone extract in postmenopausal women by proteome

  2. Analysis of the effect of a novel therapeutic for type 2 diabetes on the proteome of a muscle cell line.

    Science.gov (United States)

    Young, Pamela A; Leonard, Siobhán; Martin, Darren S D; Findlay, John B C

    2016-01-01

    Elevated serum retinol-binding protein (RBP) concentration has been implicated in the development of insulin resistance and type 2 diabetes. Two series of small molecules have been designed to lower serum levels by reducing secretion of the transthyretin-RBP complex from the liver and enhancing RBP clearance through the kidney. These small molecules were seen to improve glucose and insulin tolerance tests and to reduce body weight gain in mice rendered diabetic through a high fat diet. A proteomics study was conducted to better understand the effects of these compounds in muscle cells, muscle being the primary site for energy expenditure. One lead compound, RTC-15, is seen to have a significant effect on proteins involved in fat and glucose metabolism. This could indicate that the compound is having a direct effect on muscle tissue to improve energy homeostasis as well as a whole body effect on circulating RBP levels. This newly characterized group of antidiabetic compounds may prove useful in the treatment and prevention of insulin resistance and obesity. PMID:26573124

  3. Involvement of potential pathways in malignant transformation from Oral Leukoplakia to Oral Squamous Cell Carcinoma revealed by proteomic analysis

    Directory of Open Access Journals (Sweden)

    Li Jing

    2009-08-01

    Full Text Available Abstract Background Oral squamous cell carcinoma (OSCC is one of the most common forms of cancer associated with the presence of precancerous oral leukoplakia. Given the poor prognosis associated with oral leukoplakia, and the difficulties in distinguishing it from cancer lesions, there is an urgent need to elucidate the molecular determinants and critical signal pathways underlying the malignant transformation of precancerous to cancerous tissue, and thus to identify novel diagnostic and therapeutic target. Results We have utilized two dimensional electrophoresis (2-DE followed by ESI-Q-TOF-LC-MS/MS to identify proteins differentially expressed in six pairs of oral leukoplakia tissues with dysplasia and oral squamous cancer tissues, each pair was collected from a single patient. Approximately 85 differentially and constantly expressed proteins (> two-fold change, P Conclusion Varying levels of differentially expressed proteins were possibly involved in the malignant transformation of oral leukoplakia. Their expression levels, bioprocess, and interaction networks were analyzed using a bioinformatics approach. This study shows that the three homologs of PA28 may play an important role in malignant transformation and is an example of a systematic biology study, in which functional proteomics were constructed to help to elucidate mechanistic aspects and potential involvement of proteins. Our results provide new insights into the pathogenesis of oral cancer. These differentially expressed proteins may have utility as useful candidate markers of OSCC.

  4. The minotaur proteome

    DEFF Research Database (Denmark)

    Bunkenborg, Jakob; García, Guadalupe Espadas; Paz, Marcia Ivonne Peña;

    2010-01-01

    Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5-15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides...... from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC-MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search...

  5. Proteomic analysis of INS-1 rat insulinoma cells: ER stress effects and the protective role of exenatide, a GLP-1 receptor agonist.

    Directory of Open Access Journals (Sweden)

    Mi-Kyung Kim

    Full Text Available Beta cell death caused by endoplasmic reticulum (ER stress is a key factor aggravating type 2 diabetes. Exenatide, a glucagon-like peptide (GLP-1 receptor agonist, prevents beta cell death induced by thapsigargin, a selective inhibitor of ER calcium storage. Here, we report on our proteomic studies designed to elucidate the underlying mechanisms. We conducted comparative proteomic analyses of cellular protein profiles during thapsigargin-induced cell death in the absence and presence of exenatide in INS-1 rat insulinoma cells. Thapsigargin altered cellular proteins involved in metabolic processes and protein folding, whose alterations were variably modified by exenatide treatment. We categorized the proteins with thapsigargin initiated alterations into three groups: those whose alterations were 1 reversed by exenatide, 2 exaggerated by exenatide, and 3 unchanged by exenatide. The most significant effect of thapsigargin on INS-1 cells relevant to their apoptosis was the appearance of newly modified spots of heat shock proteins, thimet oligopeptidase and 14-3-3β, ε, and θ, and the prevention of their appearance by exenatide, suggesting that these proteins play major roles. We also found that various modifications in 14-3-3 isoforms, which precede their appearance and promote INS-1 cell death. This study provides insights into the mechanisms in ER stress-caused INS-1 cell death and its prevention by exenatide.

  6. Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Petersen, Jørgen; Nielsen, Søren Jensby; Morsczeck, Christian; Morszeck, Christian; Jensen, Peter B; Sehested, Maxwell; Grauslund, Morten

    2010-01-01

    the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 muM belinostat were analysed by 2-D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images...... HSP90B that all were related to the proto-oncogene proteins p53, Myc, activator protein 1, and c-fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be...

  7. The proteomics of quiescent and nonquiescent cell differentiation in yeast stationary-phase cultures.

    Science.gov (United States)

    Davidson, George S; Joe, Ray M; Roy, Sushmita; Meirelles, Osorio; Allen, Chris P; Wilson, Melissa R; Tapia, Phillip H; Manzanilla, Elaine E; Dodson, Anne E; Chakraborty, Swagata; Carter, Mark; Young, Susan; Edwards, Bruce; Sklar, Larry; Werner-Washburne, Margaret

    2011-04-01

    As yeast cultures enter stationary phase in rich, glucose-based medium, differentiation of two major subpopulations of cells, termed quiescent and nonquiescent, is observed. Differences in mRNA abundance between exponentially growing and stationary-phase cultures and quiescent and nonquiescent cells are known, but little was known about protein abundance in these cells. To measure protein abundance in exponential and stationary-phase cultures, the yeast GFP-fusion library (4159 strains) was examined during exponential and stationary phases, using high-throughput flow cytometry (HyperCyt). Approximately 5% of proteins in the library showed twofold or greater changes in median fluorescence intensity (abundance) between the two conditions. We examined 38 strains exhibiting two distinct fluorescence-intensity peaks in stationary phase and determined that the two fluorescence peaks distinguished quiescent and nonquiescent cells, the two major subpopulations of cells in stationary-phase cultures. GFP-fusion proteins in this group were more abundant in quiescent cells, and half were involved in mitochondrial function, consistent with the sixfold increase in respiration observed in quiescent cells and the relative absence of Cit1p:GFP in nonquiescent cells. Finally, examination of quiescent cell-specific GFP-fusion proteins revealed symmetry in protein accumulation in dividing quiescent and nonquiescent cells after glucose exhaustion, leading to a new model for the differentiation of these cells. PMID:21289090

  8. Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense (Dinophyceae.

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    Full Text Available Dinoflagellates are the major causative agents of harmful algal blooms in the coastal zone, which has resulted in adverse effects on the marine ecosystem and public health, and has become a global concern. Knowledge of cell cycle regulation in proliferating cells is essential for understanding bloom dynamics, and so this study compared the protein profiles of Prorocentrum donghaiense at different cell cycle phases and identified differentially expressed proteins using 2-D fluorescence difference gel electrophoresis combined with MALDI-TOF-TOF mass spectrometry. The results showed that the synchronized cells of P. donghaiense completed a cell cycle within 24 hours and cell division was phased with the diurnal cycle. Comparison of the protein profiles at four cell cycle phases (G1, S, early and late G2/M showed that 53 protein spots altered significantly in abundance. Among them, 41 were identified to be involved in a variety of biological processes, e.g. cell cycle and division, RNA metabolism, protein and amino acid metabolism, energy and carbon metabolism, oxidation-reduction processes, and ABC transport. The periodic expression of these proteins was critical to maintain the proper order and function of the cell cycle. This study, to our knowledge, for the first time revealed the major biological processes occurring at different cell cycle phases which provided new insights into the mechanisms regulating the cell cycle and growth of dinoflagellates.

  9. Proteomics Funding Opportunity - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    To expand the understanding of how cells sense and respond to changes in their physical environment, the NCI is seeking to perform proteomic assays on the panel of cell lines grown on a variety of substrates. These assays will provide insight into changes in protein levels or phosphorylation changes that could reflect the activity of mechano-transduction pathways.

  10. Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa.

    Science.gov (United States)

    Tipton, Jeremiah D; Tran, John C; Catherman, Adam D; Ahlf, Dorothy R; Durbin, Kenneth R; Lee, Ji Eun; Kellie, John F; Kelleher, Neil L; Hendrickson, Christopher L; Marshall, Alan G

    2012-03-01

    Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa. PMID:22356091

  11. Three Human Cell Types Respond to Multi-Walled Carbon Nanotubes and Titanium Dioxide Nanobelts with Cell-Specific Transcriptomic and Proteomic Expression Patterns.

    Energy Technology Data Exchange (ETDEWEB)

    Tilton, Susan C.; Karin, Norman J.; Tolic, Ana; Xie, Yumei; Lai, Xianyin; Hamilton, Raymond F.; Waters, Katrina M.; Holian, Andrij; Witzmann, Frank A.; Orr, Galya

    2014-08-01

    The growing use of engineered nanoparticles (NPs) in commercial and medical applications raises the urgent need for tools that can predict NP toxicity. Global transcriptome and proteome analyses were conducted on three human cell types, exposed to two high aspect ratio NP types, to identify patterns of expression that might indicate high versus low NP toxicity. Three cell types representing the most common routes of human exposure to NPs, including macrophage-like (THP-1), small airway epithelial and intestinal (Caco-2/HT29-MTX) cells, were exposed to TiO2 nanobelts (TiO2-NB; high toxicity) and multi-walled carbon nanotubes (MWCNT; low toxicity) at low (10 µg/mL) and high (100 µg/mL) concentrations for 1 and 24 h. Unique patterns of gene and protein expressions were identified for each cell type, with no differentially expressed (p < 0.05, 1.5-fold change) genes or proteins overlapping across all three cell types. While unique to each cell type, the early response was primarily independent of NP type, showing similar expression patterns in response to both TiO2-NB and MWCNT. The early response might, therefore, indicate a general response to insult. In contrast, the 24 h response was unique to each NP type. The most significantly (p < 0.05) enriched biological processes in THP-1 cells indicated TiO2-NB regulation of pathways associated with inflammation, apoptosis, cell cycle arrest, DNA replication stress and genomic instability, while MWCNT-regulated pathways indicated increased cell proliferation, DNA repair and anti-apoptosis. These two distinct sets of biological pathways might, therefore, underlie cellular responses to high and low NP toxicity, respectively.

  12. Alterations in the nuclear proteome of HIV-1 infected T-cells

    International Nuclear Information System (INIS)

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines

  13. Alterations in the nuclear proteome of HIV-1 infected T-cells

    Energy Technology Data Exchange (ETDEWEB)

    DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

    2014-11-15

    Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

  14. Proteomics: Protein Identification Using Online Databases

    Science.gov (United States)

    Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

    2012-01-01

    Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

  15. Proteome Sci.

    OpenAIRE

    Mann Matthias; Poustka Albert J; Mann Karlheinz

    2010-01-01

    Abstract Background Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. Results The present report contains the deter...

  16. Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix

    OpenAIRE

    Luecke, N; Templin, C; Muetzelburg, M V; Neumann, D.; Just, I; Pich, A.(IFIC, Universitat de València, CSIC, Apt. Correus 22085, 46071 , València, Spain)

    2010-01-01

    Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. ...

  17. Comparative proteomic analysis of differentially expressed proteins between K562 and K562/ADM cells

    Institute of Scientific and Technical Information of China (English)

    SHEN Shao-hua; GU Long-jun; LIU Pei-qing; YE Xin; CHANG Wei-shan; LI Ben-shang

    2008-01-01

    Background Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia.The resistance process is multifactorial.To realize the totaI factors involved in multidrug resistance,we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins(CRKL)using siRNA to determine its role in K562/ADM cells.Methods Altered protein expressions between K562/S(K562 ADM-sensitive cell line)and K562/ADM(K562 multidrug resistant cell line induced by adriamycin)were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile,we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis.Furthermore,we used RNA interference to silence the CRKL gene expression.Results Among the 9 differentially expressed proteins,3 were up-regulated in K562/ADM cells,while 6 were down-regulated in the K562/ADM cells compared with its parent cell line.The expression of CRKL was up-regulated significantly in K562/ADM cells,and it can be decreased by recombinant lentivirus.Moreover,the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.Conclusions The data provided the differentially expressed proteins jn K562 and jts resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer.We found CRKL may be a new protein involved in the multidrug resistanse of leukaemia cells.

  18. Proteomic and phosphoproteomic analysis of signalling by adhesion and growth factor receptors in mammary epithelial cells

    OpenAIRE

    Paul, Nikki

    2014-01-01

    Cell adhesion and communication are essential for tissue morphogenesis and repair in healthy multicellular organisms. However, dysregulation of these processes can drive disease progression in conditions such as cancer. Selective cell adhesion to the extracellular matrix is mediated by integrins, a family of transmembrane receptors that compartmentalise signalling and organise the cytoskeleton. Adhesion receptors provide spatial cues to cells to allow them to respond to growth factor and cyto...

  19. The Wolbachia WO bacteriophage proteome in the Aedes albopictus C/wStr1 cell line: evidence for lytic activity?

    Science.gov (United States)

    Baldridge, Gerald D; Markowski, Todd W; Witthuhn, Bruce A; Higgins, LeeAnn; Baldridge, Abigail S; Fallon, Ann M

    2016-01-01

    Wolbachia pipientis (Rickettsiales), an obligate intracellular alphaproteobacterium in insects, manipulates host reproduction to maximize invasion of uninfected insect populations. Modification of host population structure has potential applications for control of pest species, particularly if Wolbachia can be maintained, manipulated, and genetically engineered in vitro. Although Wolbachia maintains an obligate mutualism with genome stability in nematodes, arthropods can be co-infected with distinct Wolbachia strains, and horizontal gene transfer between strains is potentially mediated by WO phages encoded within Wolbachia genomes. Proteomic analysis of a robust, persistent infection of a mosquito cell line with wStr from the planthopper, Laodelphax striatellus, revealed expression of a full array of WO phage genes, as well as nine of ten non-phage genes that occur between two distinct clusters of WOMelB genes in the genome of wMel, which infects Drosophila melanogaster. These non-phage genes encode potential host-adaptive proteins and are expressed in wStr at higher levels than phage structural proteins. A subset of seven of the non-phage genes is flanked by highly conserved non-coding sequences, including a putative promoter element, that are not present in a syntenically arranged array of homologs in plasmids from three tick-associated Rickettsia spp. These studies expand our understanding of wStr in a host cell line derived from the mosquito, Aedes albopictus, and provide a basis for investigating conditions that favor the lytic phase of the WO phage life cycle and recovery of infectious phage particles. PMID:26427709

  20. Comparative proteomic study of nasopharyngeal carcinoma cell lines with different radiosensitivity

    International Nuclear Information System (INIS)

    Objective: To investigate the proteins which were associated with radiosensitivity of nasopharyngeal carcinoma (NPC) cells and could be used to predict the radiosensitivity. Methods: A radioresistant subclone cell line CNE-2 (R743) derived from NPC cell line CNE-2 was established. Radiosensitivity and cell cycle characteristics of CNE-2 and CNE-2 (R743) were examined and compared by clonogenic survival assay and flow cytometry. The total proteins from the two cell lines were extracted and separated by two-dimensional gel electrophoresis, and the images were analyzed by Image Master 7.0 analysis software. Differentially expressed proteins in the two cell lines were identified through MALDI-TOF/TOF peptide mass fingerprint and searched in the protein sequence database. The protein expressions were confirmed by RT-PCR and Western blot. Results: Totally seven differentially expressed proteins were identified, six of which were upregulated and one downregulated in the radioresistant CNE-2 (R743), compared with those of CNE-2. Three out of the seven, Annexin A2, Tropomyosin 4 and GRP78 were upregulated in the CNE-2 (R743), which were confirmed by Western blot and RT-PCR (t=24.22, 24.20, 29.19, P<0.05). Conclusions: Differentially expressed proteins might be involved in different radiosensitivities of nasopharyngeal carcinoma cell lines, among which Annexin A2, Tropomyosin 4 and GRP78 could be the candidate biomarkers for predicting radiosensitivity of nasopharyngeal carcinoma cells. (authors)

  1. 309 proteomic analysis of the blastocoel fluid and remaining cells of bovine blastocysts

    DEFF Research Database (Denmark)

    Jensen, P L; Groendahl, M L; Beck, Helle;

    2012-01-01

    Human embryonic stem cells (hESC) are derived from the human blastocyst and possess the potential to differentiate into any cell type present in the adult human body. Human ESC are considered to have great potential in regenerative medicine for the future treatment of severe diseases and conditions...

  2. Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

    DEFF Research Database (Denmark)

    López-Villar, Elena; Monteoliva, Lucía; Larsen, Martin Røssel;

    2006-01-01

    Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that...

  3. Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase

    Directory of Open Access Journals (Sweden)

    Deckwer Wolf-Dieter

    2006-10-01

    Full Text Available Abstract High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320, both carrying a heterologous dextransucrase (dsrS gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available. High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319, not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses. The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the

  4. Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.

    Science.gov (United States)

    Sun, Liangliang; Dubiak, Kyle M; Peuchen, Elizabeth H; Zhang, Zhenbin; Zhu, Guijie; Huber, Paul W; Dovichi, Norman J

    2016-07-01

    Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32-, and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1 400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ∼0.8 μg (16-cell embryo) to ∼0.2 μg (50-cell embryo), the number of protein group identifications declined from 1 466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32-, and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development. PMID:27314579

  5. Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

    Science.gov (United States)

    Chang, Hsin-Yi; Li, Ming-Hua; Huang, Tsui-Chin; Hsu, Chia-Lang; Tsai, Shang-Ru; Lee, Si-Chen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-02-01

    Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 μm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy. PMID:25556991

  6. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    LENUS (Irish Health Repository)

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  7. Natural taurine promotes apoptosis of human hepatic stellate cells in proteomics analysis

    OpenAIRE

    Deng, Xin; Liang, Jian; Lin, Zhi-Xiu; Wu, Fa-Sheng; Zhang, Ya-Ping; Zhang, Zhi-Wei

    2010-01-01

    AIM: To study the differential expression of proteins between natural taurine treated hepatic stellate cells and controls, and investigate the underlying regulatory mechanism of natural taurine in inhibiting hepatic fibrosis.

  8. Proteomic analysis of cell lines to identify the irinotecan resistance proteins

    Indian Academy of Sciences (India)

    Xing-Chen Peng; Feng-Ming Gong; Meng Wei; X I Chen; Y E Chen; K E Cheng; Feng Gao; Feng Xu; FENG Bi; Ji-Yan Liu

    2010-12-01

    Chemotherapeutic drug resistance is a frequent cause of treatment failure in colon cancer patients. Several mechanisms have been implicated in drug resistance. However, they are not sufficient to exhaustively account for this resistance emergence. In this study, two-dimensional gel electrophoresis (2-DE) and the PDQuest software analysis were applied to compare the differential expression of irinotecan-resistance-associated protein in human colon adenocarcinoma LoVo cells and irinotecan-resistant LoVo cells (LoVo/irinotecan). The differential protein dots were excised and analysed by ESI-Q-TOF mass spectrometry (MS). Fifteen proteins were identified, including eight proteins with decreased expression and seven proteins with increased expression. The identified known proteins included those that function in diverse biological processes such as cellular transcription, cell apoptosis, electron transport/redox regulation, cell proliferation/differentiation and retinol metabolism pathways. Identification of such proteins could allow improved understanding of the mechanisms leading to the acquisition of chemoresistance.

  9. HeLa cell response proteome alterations induced by mammalian reovirus T3D infection

    OpenAIRE

    Coombs, Kevin M.

    2013-01-01

    Background Cells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such stud...

  10. Proteomic analysis of pathways involved in estrogen-induced growth and apoptosis of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Zhang-Zhi Hu

    Full Text Available BACKGROUND: Estrogen is a known growth promoter for estrogen receptor (ER-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we sought to identify signaling networks that are triggered by estradiol (E2 in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C versus cells that proliferate upon exposure to E2 (MCF-7. The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1 is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation. CONCLUSIONS: G-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen.

  11. Mechanistic Study of the Phytocompound, 2- β -D-Glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne in Human T-Cell Acute Lymphocytic Leukemia Cells by Using Combined Differential Proteomics and Bioinformatics Approaches

    Science.gov (United States)

    Shiau, Jeng-Yuan; Yin, Shu-Yi; Chang, Shu-Lin; Hsu, Yi-Jou; Chen, Kai-Wei; Kuo, Tien-Fen; Feng, Ching-Shan; Yang, Ning-Sun; Shyur, Lie-Fen; Yang, Wen-Chin; Wen, Tuan-Nan

    2015-01-01

    Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT) can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis), methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia. PMID:26557148

  12. Mechanistic Study of the Phytocompound, 2-β-D-Glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne in Human T-Cell Acute Lymphocytic Leukemia Cells by Using Combined Differential Proteomics and Bioinformatics Approaches

    Directory of Open Access Journals (Sweden)

    Jeng-Yuan Shiau

    2015-01-01

    Full Text Available Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis, methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia.

  13. Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis.

    Directory of Open Access Journals (Sweden)

    Caroline Schrodt

    Full Text Available Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against colonization and infection. Effects of rifaximin on epithelial cells were further examined by comparing the protein expression profile of cells pretreated with rifaximin, rifampin (control antibiotic, or media (untreated. Two-dimensional (2-D gel electrophoresis identified 36 protein spots that were up- or down-regulated by over 1.7-fold in rifaximin treated cells compared to controls. 15 of these spots were down-regulated, including annexin A5, intestinal-type alkaline phosphatase, histone H4, and histone-binding protein RbbP4. 21 spots were up-regulated, including heat shock protein (HSP 90α and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. These data suggested that in addition to its antimicrobial properties, rifaximin may alter host cell physiology that provides cytoprotective effects against bacterial pathogens.

  14. Identification of Secreted Proteins from Ionizing Radiation-Induced Senescent MCF7 Cells Using Comparative Proteomics

    International Nuclear Information System (INIS)

    Cellular senescence was first described by Hayflick and Moorhead in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrest. The majority of senescent cells assume a characteristic flattened and enlarged morphological change, senescence associated β-galactosidase positivity and over the years a large number of molecular phenotypes have been described, such as changes in gene expression, protein processing and chromatin organization. In contrast to apoptosis, senescence does not destroy the cells but leaves them metabolically and synthetically active and therefore able to affect their microenvironment. In particular, senescent fibroblasts and some cancer cells were found to secrete proteins with known or putative tumor-promoting functions such as growth factors or proteolytic enzymes. However, the knowledge about secreted proteins from senescent tumor cells and their functions to surrounding cells is still lacking. In this study, changes of senescence-associated secretory protein expression profile were observed in MCF7 human breast cancer cells exposed to gamma-ray radiation using two dimensional electrophoresis. Also, we identified up-regulated secretory proteins during ionizing radiation-induced cellular senescence

  15. Identification of Secreted Proteins from Ionizing Radiation-Induced Senescent MCF7 Cells Using Comparative Proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Han, Na Kyung; Kim, Han Na; Hong, Mi Na; Park, Su Min; Lee, Jae Seon [Korea Institue of Radiological and Medical Sciences, Seoul (Korea, Republic of); Chi, Seong Gil [Korea University, Seoul (Korea, Republic of)

    2010-05-15

    Cellular senescence was first described by Hayflick and Moorhead in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrest. The majority of senescent cells assume a characteristic flattened and enlarged morphological change, senescence associated beta-galactosidase positivity and over the years a large number of molecular phenotypes have been described, such as changes in gene expression, protein processing and chromatin organization. In contrast to apoptosis, senescence does not destroy the cells but leaves them metabolically and synthetically active and therefore able to affect their microenvironment. In particular, senescent fibroblasts and some cancer cells were found to secrete proteins with known or putative tumor-promoting functions such as growth factors or proteolytic enzymes. However, the knowledge about secreted proteins from senescent tumor cells and their functions to surrounding cells is still lacking. In this study, changes of senescence-associated secretory protein expression profile were observed in MCF7 human breast cancer cells exposed to gamma-ray radiation using two dimensional electrophoresis. Also, we identified up-regulated secretory proteins during ionizing radiation-induced cellular senescence

  16. Perspectives for computational modeling of cell replacement for neurological disorders

    Directory of Open Access Journals (Sweden)

    James B Aimone

    2013-11-01

    Full Text Available Mathematical modeling of anatomically-constrained neural networks has provided significant insights regarding the response of networks to neurological disorders or injury. A logical extension of these models is to incorporate treatment regimens to investigate network responses to intervention. The addition of nascent neurons from stem cell precursors into damaged or diseased tissue has been used as a successful therapeutic tool in recent decades. Interestingly, models have been developed to examine the incorporation of new neurons into intact adult structures, particularly the dentate granule neurons of the hippocampus. These studies suggest that the unique properties of maturing neurons can impact circuit behavior in unanticipated ways. In this perspective, we review the current status of models used to examine damaged CNS structures with particular focus on cortical damage due to stroke. Secondly, we suggest that computational modeling of cell replacement therapies can be made feasible by implementing approaches taken by current models of adult neurogenesis. The development of these models is critical for generating hypotheses regarding transplant therapies and improving outcomes by tailoring transplants to desired effects.

  17. Proteomic analysis of mechanisms of hypoxia-induced apoptosis in trophoblastic cells

    Directory of Open Access Journals (Sweden)

    Shin-ichi Ishioka, Yoshiaki Ezaka, Kota Umemura, Takuhiro Hayashi, Toshiaki Endo, Tsuyoshi Saito

    2007-01-01

    Full Text Available Preeclampsia is often accompanied by hypoxia of the placenta and this condition induces apoptosis in trophoblastic cells. The aim of this study was to characterize global changes of apoptosis-related proteins induced by hypoxia in trophoblastic cells so as to clarify the mechanism of hypoxia-induced apoptosis by using the PoweBlot, an antibody-based Western array. Human choriocarcinoma cell line JAR was cultured for 24 hours under aerobic and hypoxic conditions. Hypoxia induced apoptosis accompanied by increased expression of Bcl-x, Caspase-3 and -9, Hsp70, PTEN, and Bag-1. Bad, pan-JNK/SAPK-1, Bcl-2, Bid, and Caspase-8 showed decreased expression. Hypoxia-induced apoptosis was increased with the transfection of a bag-1 antisense oligonucleotide. The bag-1 antisense oligonucleotide affected the expression of Bid, Bad, Bcl-2, JNK, and phosphorylated JNK, although expression of PTEN and Bcl-X did not change. Bag-1 may inhibit apoptosis by suppressing the expression of Bid and Bad. It may also enhance apoptosis by inhibiting the expression of Bcl-2 and by modulating phosphorylation of JNK. Both mitochondrial and stress-activated apoptosis pathways played important roles in the hypoxia induced cell death of trophoblastic cells. These findings will contribute to establish new approach to detect hypoxic stress of the placenta, which leads to preeclampsia and other hypoxia-related obstetrics complications.

  18. Visualizing Meta-Features in Proteomic Maps

    Directory of Open Access Journals (Sweden)

    Lepouras George

    2011-07-01

    Full Text Available Abstract Background The steps of a high-throughput proteomics experiment include the separation, differential expression and mass spectrometry-based identification of proteins. However, the last and more challenging step is inferring the biological role of the identified proteins through their association with interaction networks, biological pathways, analysis of the effect of post-translational modifications, and other protein-related information. Results In this paper, we present an integrative visualization methodology that allows combining experimentally produced proteomic features with protein meta-features, typically coming from meta-analysis tools and databases, in synthetic Proteomic Feature Maps. Using three proteomics analysis scenarios, we show that the proposed visualization approach is effective in filtering, navigating and interacting with the proteomics data in order to address visually challenging biological questions. The novelty of our approach lies in the ease of integration of any user-defined proteomic features in easy-to-comprehend visual representations that resemble the familiar 2D-gel images, and can be adapted to the user's needs. The main capabilities of the developed VIP software, which implements the presented visualization methodology, are also highlighted and discussed. Conclusions By using this visualization and the associated VIP software, researchers can explore a complex heterogeneous proteomics dataset from different perspectives in order to address visually important biological queries and formulate new hypotheses for further investigation. VIP is freely available at http://pelopas.uop.gr/~egian/VIP/index.html.

  19. Legume proteomics: Progress, prospects, and challenges.

    Science.gov (United States)

    Rathi, Divya; Gayen, Dipak; Gayali, Saurabh; Chakraborty, Subhra; Chakraborty, Niranjan

    2016-01-01

    Legumes are the major sources of food and fodder with strong commercial relevance, and are essential components of agricultural ecosystems owing to their ability to carry out endosymbiotic nitrogen fixation. In recent years, legumes have become one of the major choices of plant research. The legume proteomics is currently represented by more than 100 reference maps and an equal number of stress-responsive proteomes. Among the 48 legumes in the protein databases, most proteomic studies have been accomplished in two model legumes, soybean, and barrel medic. This review highlights recent contributions in the field of legume proteomics to comprehend the defence and regulatory mechanisms during development and adaptation to climatic changes. Here, we attempted to provide a concise overview of the progress in legume proteomics and discuss future developments in three broad perspectives: (i) proteome of organs/tissues; (ii) subcellular compartments; and (iii) spatiotemporal changes in response to stress. Such data mining may aid in discovering potential biomarkers for plant growth, in general, apart from essential components involved in stress tolerance. The prospect of integrating proteome data with genome information from legumes will provide exciting opportunities for plant biologists to achieve long-term goals of crop improvement and sustainable agriculture. PMID:26563903

  20. Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways.

    Science.gov (United States)

    Govey, Peter M; Jacobs, Jon M; Tilton, Susan C; Loiselle, Alayna E; Zhang, Yue; Freeman, Willard M; Waters, Katrina M; Karin, Norman J; Donahue, Henry J

    2014-06-01

    Osteocytes, positioned within bone׳s porous structure, are subject to interstitial fluid flow upon whole bone loading. Such fluid flow is widely theorized to be a mechanical signal transduced by osteocytes, initiating a poorly understood cascade of signaling events mediating bone adaptation to mechanical load. The objective of this study was to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels using high-throughput approaches. Osteocyte-like MLO-Y4 cells were subjected to 2h of oscillating fluid flow (1Pa peak shear stress) and analyzed following 0, 2, 8, and 24h post-flow incubation. Transcriptomic microarray analysis, followed by gene ontology pathway analysis, demonstrated fluid flow regulation of genes consistent with both known and unknown metabolic and inflammatory responses in bone. Additionally, two of the more highly up-regulated gene products - chemokines Cxcl1 and Cxcl2, supported by qPCR - have not previously been reported as responsive to fluid flow. Proteomic analysis demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. Finally, an integrative pathway analysis merging fold changes in transcript and protein levels predicted signaling nodes not directly detected at the sampled time points, including transcription factors c-Myc, c-Jun, and RelA/NF-κB. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as possible paracrine agents for osteoblastic and osteoclastic recruitment. Moreover, these results demonstrate the utility of integrative, high-throughput approaches in place of a traditional candidate approach for identifying novel mechano-sensitive signaling molecules. PMID:24720889

  1. A cell-free protein synthesis system for high-throughput proteomics

    OpenAIRE

    Sawasaki, Tatsuya; Ogasawara, Tomio; Morishita, Ryo; Endo, Yaeta

    2002-01-01

    We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many ...

  2. Effects of Stratospheric Conditions on the Viability, Metabolism and Proteome of Prokaryotic Cells

    Directory of Open Access Journals (Sweden)

    Dagmar Chudobova

    2015-08-01

    Full Text Available The application of ultraviolet (UV radiation to inhibit bacterial growth is based on the principle that the exposure of DNA to UV radiation results in the formation of cytotoxic lesions, leading to inactivation of microorganisms. Herein, we present the impacts of UV radiation on bacterial cultures’ properties from the biological, biochemical and molecular biological perspective. For experiments, commercial bacterial cultures (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli and Salmonella typhimurium and isolates from patients with bacterial infections (Proteus mirabilis and Pseudomonas aeruginosa were employed. The above-mentioned strains were exposed to UV using a laboratory source and to stratospheric UV using a 3D printed probe carried by a stratospheric balloon. The length of flight was approximately two hours, and the probe was enriched by sensors for the external environment (temperature, pressure and relative humidity. After the landing, bacterial cultures were cultivated immediately. Experimental results showed a significant effect of UV radiation (both laboratory UV and UV from the stratosphere on the growth, reproduction, behavior and structure of bacterial cultures. In all parts of the experiment, UV from the stratosphere showed stronger effects when compared to the effects of laboratory UV. The growth of bacteria was inhibited by more than 50% in all cases; moreover, in the case of P. aeruginosa, the growth was even totally inhibited. Due to the effect of UV radiation, an increased susceptibility of bacterial strains to environmental influences was also observed. By using commercial tests for biochemical markers of Gram-positive and Gram-negative strains, significant disparities in exposed and non-exposed strains were found. Protein patterns obtained using MALDI-TOF mass spectrometry revealed that UV exposure is able to affect the proteins’ expression, leading to their downregulation, observed

  3. A proteomic analysis of mushroom polysaccharide-treated HepG2 cells.

    Science.gov (United States)

    Chai, Yangyang; Wang, Guibin; Fan, Lili; Zhao, Min

    2016-01-01

    The anti-tumor properties of fungal polysaccharides have gained significant recognition in Asia and tropical America. In this study, the differential expression of proteins in normal HepG2 cells and those treated with polysaccharides that had been isolated from Phellinus linteus (PL), Ganoderma lucidum (GL) and Auricularia auricula (AA) was investigated. Using two-dimensional electrophoresis (2DE), a total of 104 protein spots were determined to be overexpressed in these cells compared with noncancerous regions. A total of 59 differentially expressed proteins were identified through MALDI-TOF-MS. In addition, 400 biological processes (BP), 133 cell components (CC) and 146 molecular functions (MF) were enriched by Gene Ontology (GO) analysis, and 78 KEGG pathways were enriched by pathway enrichment. Protein-Protein Interaction (PPI) analysis demonstrated the interaction networks affected by polysaccharides in HepG2 cells. Then, DJ-1 and 14-3-3 were identified as the key proteins in the networks, and the expression of the mRNA and proteins were evaluated using Real-time quantitative PCR (qRT-PCR) and Western blotting (WB), respectively. The results were in agreement with the 2DE. These results provided information on significant proteins of hepatocellular carcinoma (HCC) and form an important basis for the future development of valuable medicinal mushroom resources. PMID:27020667

  4. H Ferritin Gene Silencing in a Human Metastatic Melanoma Cell Line: A Proteomic Analysis

    DEFF Research Database (Denmark)

    Di Sanzo, Maddalena; Gaspari, Marco; Misaggi, Roberta; Romeo, Francesco; Falbo, Lucia; De Marco, Carmela; Agosti, Valter; Quaresima, Barbara; Barni, Tullio; Viglietto, Giuseppe; Larsen, Martin Røssel; Cuda, Giovanni; Costanzo, Francesco; Faniello, Maria Concetta

    2011-01-01

    Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation and ...

  5. Proteome analysis of butyrate-treated chronic myelogenous leukemia K562 cells

    Czech Academy of Sciences Publication Activity Database

    Halada, Petr; Grebeňová, D.; Pešlová, G.; Havlíček, Vladimír; Hrkal, Z.

    Edinburgh, 2003, s. -. [International Mass Spectrometry Conference /16./. Edingurgh (GB), 31.08.2003-05.09.2003] R&D Projects: GA ČR GA303/01/1445; GA MZd NL7681 Institutional research plan: CEZ:AV0Z5020903 Keywords : butyrate-treated * leukemie k562 * cells Subject RIV: EE - Microbiology, Virology

  6. The proteomic study of sodium butyrate antiproliferative/cytodifferentiation effects on K562 cells

    Czech Academy of Sciences Publication Activity Database

    Grebeňová, D.; Kuželová, K.; Pluskalová, M.; Pešlová, G.; Halada, Petr; Hrkal, Z.

    2006-01-01

    Roč. 37, - (2006), s. 210-217. ISSN 1079-9796 R&D Projects: GA MZd NL7681 Institutional research plan: CEZ:AV0Z50200510 Keywords : k562 * cell cycle * hemoglobin synthesis Subject RIV: EE - Microbiology, Virology Impact factor: 2.678, year: 2006

  7. Proteome array identification of bioactive soluble proteins/peptides in matrigel; relevance to stem cell responses

    Science.gov (United States)

    Matrigel and similar commercial products are extracts of the Engelbreth-Holm-Swarm sarcoma that provide a basement-membrane-like attachment factor or gel that is used to grow cells on or in. To ascertain further what proteins may be present in Matrigel, besides its major basement-membrane constitue...

  8. Proteomic Biomarkers of Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Natacha Diaz-Prieto

    2008-01-01

    Full Text Available Biomarkers provide a powerful approach to understanding the spectrum of cardiovascular diseases. They have application in screening, diagnostic, prognostication, prediction of recurrences and monitoring of therapy. The “omics” tool are becoming very useful in the development of new biomarkers in cardiovascular diseases. Among them, proteomics is especially fitted to look for new proteins in health and disease and is playing a significant role in the development of new diagnostic tools in cardiovascular diagnosis and prognosis. This review provides an overview of progress in applying proteomics to atherosclerosis. First, we describe novel proteins identified analysing atherosclerotic plaques directly. Careful analysis of proteins within the atherosclerotic vascular tissue can provide a repertoire of proteins involved in vascular remodelling and atherogenesis. Second, we discuss recent data concerning proteins secreted by atherosclerotic plaques. The definition of the atheroma plaque secretome resides in that proteins secreted by arteries can be very good candidates of novel biomarkers. Finally we describe proteins that have been differentially expressed (versus controls by individual cells which constitute atheroma plaques (endothelial cells, vascular smooth muscle cells, macrophages and foam cells as well as by circulating cells (monocytes, platelets or novel biomarkers present in plasma.

  9. Proteomic Biomarkers of Atherosclerosis.

    Science.gov (United States)

    Vivanco, F; Padial, L R; Darde, V M; de la Cuesta, F; Alvarez-Llamas, G; Diaz-Prieto, Natacha; Barderas, M G

    2008-01-01

    SUMMARY: Biomarkers provide a powerful approach to understanding the spectrum of cardiovascular diseases. They have application in screening, diagnostic, prognostication, prediction of recurrences and monitoring of therapy. The "omics" tool are becoming very useful in the development of new biomarkers in cardiovascular diseases. Among them, proteomics is especially fitted to look for new proteins in health and disease and is playing a significant role in the development of new diagnostic tools in cardiovascular diagnosis and prognosis. This review provides an overview of progress in applying proteomics to atherosclerosis. First, we describe novel proteins identified analysing atherosclerotic plaques directly. Careful analysis of proteins within the atherosclerotic vascular tissue can provide a repertoire of proteins involved in vascular remodelling and atherogenesis. Second, we discuss recent data concerning proteins secreted by atherosclerotic plaques. The definition of the atheroma plaque secretome resides in that proteins secreted by arteries can be very good candidates of novel biomarkers. Finally we describe proteins that have been differentially expressed (versus controls) by individual cells which constitute atheroma plaques (endothelial cells, vascular smooth muscle cells, macrophages and foam cells) as well as by circulating cells (monocytes, platelets) or novel biomarkers present in plasma. PMID:19578499

  10. Exposure to cobalt causes transcriptomic and proteomic changes in two rat liver derived cell lines.

    Directory of Open Access Journals (Sweden)

    Matthew G Permenter

    Full Text Available Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.

  11. Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix.

    Science.gov (United States)

    Luecke, Nina; Templin, Christian; Muetzelburg, Marika Victoria; Neumann, Detlef; Just, Ingo; Pich, Andreas

    2010-03-15

    Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration. PMID:20127908

  12. Proteomic Retrieval from Nucleic Acid Depleted Space-Flown Human Cells

    Science.gov (United States)

    Hammond, D. K.; Elliott, T. F.; Holubec, K.; Baker, T. L.; Allen, P. L.; Hammond, T. G.; Love, J. E.

    2006-01-01

    Compared to experiments utilizing humans in microgravity, cell-based approaches to questions about subsystems of the human system afford multiple advantages, such as crew safety and the ability to achieve statistical significance. To maximize the science return from flight samples, an optimized method was developed to recover protein from samples depleted of nucleic acid. This technique allows multiple analyses on a single cellular sample and when applied to future cellular investigations could accelerate solutions to significant biomedical barriers to human space exploration. Cell cultures grown in American Fluoroseal bags were treated with an RNA stabilizing agent (RNAlater - Ambion), which enabled both RNA and immunoreactive protein analyses. RNA was purified using an RNAqueous(registered TradeMark) kit (Ambion) and the remaining RNA free supernatant was precipitated with 5% trichloroacetic acid. The precipitate was dissolved in SDS running buffer and tested for protein content using a bicinchoninic acid assay (1) (Sigma). Equal loads of protein were placed on SDS-PAGE gels and either stained with CyproOrange (Amersham) or transferred using Western Blotting techniques (2,3,4). Protein recovered from RNAlater-treated cells and stained with protein stain, was measured using Imagequant volume measurements for rectangles of equal size. BSA treated in this way gave quantitative data over the protein range used (Fig 1). Human renal cortical epithelial (HRCE) cells (5,6,7) grown onboard the International Space Station (ISS) during Increment 3 and in ground control cultures exhibited similar immunoreactivity profiles for antibodies to the Vitamin D receptor (VDR) (Fig 2), the beta isoform of protein kinase C (PKC ) (Fig 3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Fig 4). Parallel immunohistochemical studies on formalin-fixed flight and ground control cultures also showed positive immunostaining for VDR and other biomarkers (Fig 5). These results are

  13. Quantitative definition and monitoring of the host cell protein proteome using iTRAQ - a study of an industrial mAb producing CHO-S cell line.

    Science.gov (United States)

    Chiverton, Lesley M; Evans, Caroline; Pandhal, Jagroop; Landels, Andrew R; Rees, Byron J; Levison, Peter R; Wright, Phillip C; Smales, C Mark

    2016-08-01

    There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs. PMID:27214759

  14. In-Depth Proteomic Quantification of Cell Secretome in Serum-Containing Conditioned Medium.

    Science.gov (United States)

    Weng, Yejing; Sui, Zhigang; Shan, Yichu; Jiang, Hao; Zhou, Yuan; Zhu, Xudong; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-01

    Secreted proteins play key roles during cellular communication, proliferation, and migration. The comprehensive profiling of secreted proteins in serum-containing culture media is technically challenging. Most studies have been performed under serum-free conditions. However, these conditions might alter the status of the cells. Herein, we describe an efficient strategy that avoids the disturbance of serum by combining metabolic labeling, protein "equalization," protein fractionation, and filter-aided sample preparation, called MLEFF, enabling the identification of 534 secreted proteins from HeLa conditioned media, including 31 cytokines, and growth factors. This MLEFF strategy was also successfully applied during a comparative secretome analysis of two human hepatocellular carcinoma cell lines with differentially metastatic potentials, enabling the quantification of 61 significantly changed proteins involved in tumor invasion and metastasis. PMID:27042867

  15. Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

    Science.gov (United States)

    Sheta, Razan; Roux-Dalvai, Florence; Woo, Christina M; Fournier, Frédéric; Bourassa, Sylvie; Bertozzi, Carolyn R; Droit, Arnaud; Bachvarov, Dimcho

    2016-09-01

    This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets. PMID:27331112

  16. Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive control

    DEFF Research Database (Denmark)

    Solis, Nestor; Larsen, Martin Røssel; Cordwell, Stuart J

    2010-01-01

    lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific......0050 (pls) and penicillin-binding protein 2' (mecA), as well as bifunctional autolysin and the extracellular matrix-binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface-exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus....... that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital-acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface "shaving" technique...

  17. Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse.

    Directory of Open Access Journals (Sweden)

    Fekrije Selimi

    2009-04-01

    Full Text Available Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKgamma, are major unrecognized components of this synapse type. We demonstrate that MRCKgamma can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types.

  18. 草酸铂处理人结肠癌细胞蛋白质组分析%Proteome analysis of human colon cancer cell line treated by oxaliplatin

    Institute of Scientific and Technical Information of China (English)

    陈家诚; 吴静; 钟亮玉; 郭伟; 陈远光

    2010-01-01

    目的 分析草酸铂处理后人结肠癌Lovo细胞系的差异表达蛋白,从蛋白质组学角度探讨草酸铂的作用机制.方法 Lovo细胞系按完全随机法分2组,即实验组(含草酸铂培养液培养)和对照组(无草酸铂培养液培养).培养后收集细胞依次进行二维电泳分离并选择差异点,采用质谱分析鉴定差异蛋白.结果 两组双向电泳图谱分辨率高、重复性好,匹配率分别为80.16%(812/1013)和81.19%(829/1021).获取重复差异点22个,质谱分析其中10个,成功鉴定出与结肠癌相关6种蛋白:β2微球蛋白、剪接因子SFRS3、尿嘧啶DNA糖基化酶表达上调;60S核糖体蛋白P2、甘油醛3磷酸脱氢酶、鸟苷酸结合蛋白β2多肽样1表达下调.结论 草酸铂作用人结肠癌Lovo细胞系后有多种差异蛋白表达,其与免疫增强、pre-mRNA转录修饰、DNA损伤修复、调控蛋白翻译及翻译后加工、能量代谢及信号传导通路等有关.%Objective To analyze differential expression proteins of human colon cancer Lovo cell line treated by oxaliplatin, and to explore the antitumor mechanism of oxaliplatin in the view of proteomics perspective. Methods The Lovo cells were randomly divided into two groups, including the experimental group (cultured in oxaliplatin- containing solution) and the control group (cultured in oxaliplatin-free solution). After culture, the both groups were processed by two-dimensional gel electrophoresis to select disparates, and differential proteins were identified by mass spectrometry analyses. Results High resolution and good reproducibility were observed on electrophoretograms of both groups, with matching rates at 80.16% (812/1013) and 81.19% (829/1021) respectively. Twenty-two repeated disparate points were obtained, ten of them were selected for mass spectrometry analyses. Six colorectal cancer related proteins were successfully identified, including 3 up-regulated proteins (β-2 microglobulin, splicing factor SFRS

  19. Rifaximin-Mediated Changes to the Epithelial Cell Proteome: 2-D Gel Analysis

    OpenAIRE

    Schrodt, Caroline; McHugh, Erin E.; Gawinowicz, Mary Ann; Herbert L. DuPont; Brown, Eric L.

    2013-01-01

    Rifaximin is a semi-synthetic rifamycin derivative that is used to treat different conditions including bacterial diarrhea and hepatic encephalopathy. Rifaximin is of particular interest because it is poorly adsorbed in the intestines and has minimal effect on colonic microflora. We previously demonstrated that rifaximin affected epithelial cell physiology by altering infectivity by enteric pathogens and baseline inflammation suggesting that rifaximin conferred cytoprotection against coloniza...

  20. Comprehensive proteomic analysis of Trypanosoma cruzi epimastigote cell surface proteins by two complementary methods

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sébastien; Motta, Flávia N; Santana, Jaime M; Roepstorff, Peter; Ricart, Carlos A O

    2013-01-01

    Trypanosoma cruzi is a protozoan that causes Chagas' disease, a neglected infectious illness that affects millions of people, mostly in Latin America. Here, the cell surface subproteome of the T. cruzi epimastigote life form was characterized. In order to prepare samples enriched in epimastigote...... of the labeled proteins. Both T. cruzi subproteomes were analyzed by LC-MS/MS. The results showed that the methodologies offered comprehensive and complementary information about the parasite's plasma membrane subproteome....

  1. Neuropeptidomic Components Generated by Proteomic Functions in Secretory Vesicles for Cell–Cell Communication

    OpenAIRE

    Hook, Vivian; Bark, Steven; Gupta, Nitin; Lortie, Mark; Lu, Weiya D.; Bandeira, Nuno; Funkelstein, Lydiane; Wegrzyn, Jill; O’Connor, Daniel T.; Pevzner, Pavel

    2010-01-01

    Diverse neuropeptides participate in cell–cell communication to coordinate neuronal and endocrine regulation of physiological processes in health and disease. Neuropeptides are short peptides ranging in length from ~3 to 40 amino acid residues that are involved in biological functions of pain, stress, obesity, hypertension, mental disorders, cancer, and numerous health conditions. The unique neuropeptide sequences define their specific biological actions. Significantly, this review article di...

  2. Analysis of the immune response of human dendritic cells to Mycobacterium tuberculosis by quantitative proteomics

    OpenAIRE

    Kuo, Chiu-Ping; Chang, Kuo-Song; Hsu, Jue-Liang; Tsai, I-Fang; Lin, Andrew Boyd; Wei, Tsai-Yin; Wu, Chien-Liang; Lu, Yen-Ta

    2016-01-01

    Background The cellular immune response for Mycobacterium tuberculosis (M. tuberculosis) infection remained incompletely understood. To uncover membrane proteins involved in this infection mechanism, an integrated approach consisting of an organic solvent-assisted membrane protein digestion, stable-isotope dimethyl labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to comparatively profile the membrane protein expression of human dendritic cells upon heat...

  3. Proteomic Pathway Analysis Reveals Inflammation Increases Myeloid-Derived Suppressor Cell Resistance to Apoptosis*

    OpenAIRE

    Chornoguz, Olesya; Grmai, Lydia; Sinha, Pratima; Artemenko, Konstantin A.; Zubarev, Roman A.; Ostrand-Rosenberg, Suzanne

    2010-01-01

    Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in...

  4. Proteomic Analysis of Terminalia chebula Extract-Dependent Changes in Human Lymphoblastic T Cell Protein Expression

    OpenAIRE

    Das, Nando Dulal; Jung, Kyoung Hwa; Park, Ji Hyun; Choi, Mi Ran; LEE, HYUNG TAE; Kim, Moo Sung; Lee, Sang Rin; Chai, Young Gyu

    2012-01-01

    Terminalia chebula is a native plant from southern Asia to southwestern China that is used in traditional medicine for the treatment of malignant tumors and diabetes. This plant also has antibacterial and immunomodulatory properties. The present study assessed T. chebula extract-dependent protein expression changes in Jurkat cells. Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry and Ingenuity Pathways Analysis (IPA) were performed to assess protein expression and ...

  5. A proteomic approach to studying the differentiation of neural stem cells

    Czech Academy of Sciences Publication Activity Database

    Skalníková, Helena; Halada, Petr; Vodička, Petr; Motlík, Jan; Řehulka, Pavel; Horning, O.; Chmelík, Josef; Norregaard Jensen, O.; Kovářová, Hana

    2007-01-01

    Roč. 7, 11 (2007), s. 1825-1838. ISSN 1615-9853 R&D Projects: GA MŠk 1M0538; GA MŠk LC545 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510; CEZ:AV0Z40310501 Keywords : differentiation * neural stem cells * two-dimensional gel electrophoresis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.479, year: 2007

  6. MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

    DEFF Research Database (Denmark)

    Zhang, Yanling; Zhang, Yong; Adachi, Jun;

    2007-01-01

    Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several...... body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and......://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic...

  7. Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach.

    Science.gov (United States)

    Li, Qingdi Quentin; Hao, Jian-Jiang; Zhang, Zheng; Hsu, Iawen; Liu, Yi; Tao, Zhen; Lewi, Keidren; Metwalli, Adam R; Agarwal, Piyush K

    2016-06-01

    The Cancer Genome Atlas (TCGA) project recently identified the importance of mutations in chromatin remodeling genes in human carcinomas. These findings imply that epigenetic modulators might have a therapeutic role in urothelial cancers. To exploit histone deacetylases (HDACs) as targets for cancer therapy, we investigated the HDAC inhibitors (HDACIs) romidepsin, trichostatin A, and vorinostat as potential chemotherapeutic agents for bladder cancer. We demonstrate that the three HDACIs suppressed cell growth and induced cell death in the bladder cancer cell line 5637. To identify potential mechanisms associated with the anti-proliferative and cytotoxic effects of the HDACIs, we used quantitative proteomics to determine the proteins potentially involved in these processes. Our proteome studies identified a total of 6003 unique proteins. Of these, 2472 proteins were upregulated and 2049 proteins were downregulated in response to HDACI exposure compared to the untreated controls (P<0.05). Bioinformatic analysis further revealed that those differentially expressed proteins were involved in multiple biological functions and enzyme-regulated pathways, including cell cycle progression, apoptosis, autophagy, free radical generation and DNA damage repair. HDACIs also altered the acetylation status of histones and non-histone proteins, as well as the levels of chromatin modification proteins, suggesting that HDACIs exert multiple cytotoxic actions in bladder cancer cells by inhibiting HDAC activity or altering the structure of chromatin. We conclude that HDACIs are effective in the inhibition of cell proliferation and the induction of apoptosis in the 5637 bladder cancer cells through multiple cell death-associated pathways. These observations support the notion that HDACIs provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration. PMID:27082124

  8. Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

    Science.gov (United States)

    Ito, Hiromi; Kaji, Hiroyuki; Togayachi, Akira; Azadi, Parastoo; Ishihara, Mayumi; Geyer, Rudolf; Galuska, Christina; Geyer, Hildegard; Kakehi, Kazuaki; Kinoshita, Mitsuhiro; Karlsson, Niclas G; Jin, Chunsheng; Kato, Koichi; Yagi, Hirokazu; Kondo, Sachiko; Kawasaki, Nana; Hashii, Noritaka; Kolarich, Daniel; Stavenhagen, Kathrin; Packer, Nicolle H; Thaysen-Andersen, Morten; Nakano, Miyako; Taniguchi, Naoyuki; Kurimoto, Ayako; Wada, Yoshinao; Tajiri, Michiko; Yang, Pengyuan; Cao, Weiqian; Li, Hong; Rudd, Pauline M; Narimatsu, Hisashi

    2016-06-01

    The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples. PMID:26511985

  9. Evaluation of Three Protein-Extraction Methods for Proteome Analysis of Maize Leaf Midrib, a Compound Tissue Rich in Sclerenchyma Cells.

    Science.gov (United States)

    Wang, Ning; Wu, Xiaolin; Ku, Lixia; Chen, Yanhui; Wang, Wei

    2016-01-01

    Leaf morphology is closely related to the growth and development of maize (Zea mays L.) plants and final kernel production. As an important part of the maize leaf, the midrib holds leaf blades in the aerial position for maximum sunlight capture. Leaf midribs of adult plants contain substantial sclerenchyma cells with heavily thickened and lignified secondary walls and have a high amount of phenolics, making protein extraction and proteome analysis difficult in leaf midrib tissue. In the present study, three protein-extraction methods that are commonly used in plant proteomics, i.e., phenol extraction, TCA/acetone extraction, and TCA/acetone/phenol extraction, were qualitatively and quantitatively evaluated based on 2DE maps and MS/MS analysis using the midribs of the 10th newly expanded leaves of maize plants. Microscopy revealed the existence of substantial amounts of sclerenchyma underneath maize midrib epidermises (particularly abaxial epidermises). The spot-number order obtained via 2DE mapping was as follows: phenol extraction (655) > TCA/acetone extraction (589) > TCA/acetone/phenol extraction (545). MS/MS analysis identified a total of 17 spots that exhibited 2-fold changes in abundance among the three methods (using phenol extraction as a control). Sixteen of the proteins identified were hydrophilic, with GRAVY values ranging from -0.026 to -0.487. For all three methods, we were able to obtain high-quality protein samples and good 2DE maps for the maize leaf midrib. However, phenol extraction produced a better 2DE map with greater resolution between spots, and TCA/acetone extraction produced higher protein yields. Thus, this paper includes a discussion regarding the possible reasons for differential protein extraction among the three methods. This study provides useful information that can be used to select suitable protein extraction methods for the proteome analysis of recalcitrant plant tissues that are rich in sclerenchyma cells. PMID:27379139

  10. Fluvastatin mediated breast cancer cell death: a proteomic approach to identify differentially regulated proteins in MDA-MB-231 cells.

    Directory of Open Access Journals (Sweden)

    Anantha Koteswararao Kanugula

    Full Text Available Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death.

  11. Phenotypic and Proteomic Characteristics of Human Dental Pulp Derived Mesenchymal Stem Cells from a Natal, an Exfoliated Deciduous, and an Impacted Third Molar Tooth

    Directory of Open Access Journals (Sweden)

    Gurler Akpinar

    2014-01-01

    Full Text Available The level of heterogeneity among the isolated stem cells makes them less valuable for clinical use. The purpose of this study was to understand the level of heterogeneity among human dental pulp derived mesenchymal stem cells by using basic cell biology and proteomic approaches. The cells were isolated from a natal (NDPSCs, an exfoliated deciduous (stem cells from human exfoliated deciduous (SHED, and an impacted third molar (DPSCs tooth of three different donors. All three stem cells displayed similar features related to morphology, proliferation rates, expression of various cell surface markers, and differentiation potentials into adipocytes, osteocytes, and chondrocytes. Furthermore, using 2DE approach coupled with MALDI-TOF/TOF, we have generated a common 2DE profile for all three stem cells. We found that 62.3±7% of the protein spots were conserved among the three mesenchymal stem cell lines. Sixty-one of these conserved spots were identified by MALDI-TOF/TOF analysis. Classification of the identified proteins based on biological function revealed that structurally important proteins and proteins that are involved in protein folding machinery are predominantly expressed by all three stem cell lines. Some of these proteins may hold importance in understanding specific properties of human dental pulp derived mesenchymal stem cells.

  12. Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

    Directory of Open Access Journals (Sweden)

    Soderblom Erik J

    2013-01-01

    Full Text Available Abstract Background In sickle cell disease (SCD, the mitogen-activated protein kinase (MAPK ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs. ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown. Results To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2

  13. Quantitative proteomics of Chlorobaculum tepidum

    DEFF Research Database (Denmark)

    Falkenby, Lasse Gaarde; Szymanska, Monika; Holkenbrink, Carina;

    2011-01-01

    Chlorobaculum (Cba.) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. To gain insight into the sulfur metabolism, the proteome of Cba. tepidum cells sampled under different growth conditions has been quantified using a rapid gel...

  14. Quantitative and comparative proteomics analysis of radiosensitivity for colorectal cancer cells

    International Nuclear Information System (INIS)

    To identify the related proteins about radiosensitivity (RS) of colorectal cancers,as well as make the personal therapeutics for patients, the specimen of transplanted tumor raised by the colorectal cancer cells, LOVO and SW480 with different radiosensitivity, were examined by the fluorescence differential in-gel electrophoresis (DIGE) after labeled with CyDye DIGE fluors Cy3, Cy5 and Cy2. As the result, intensity changes of 35 spots were detected with statistical significance. 18 protein spots of them are up-regulated in tumor specimen of LOVO, otherwise 17 are up-regulated in specimen of SW480. And 27 of them were identified by MALDI-TOF-MS successfully including 17 in LOVO and 15 in SW480. These differential expressed proteins may become the mocular markers and have a key role in predicting the RS of colorectal cancer. (authors)

  15. Scanning the cell surface proteome of cancer cells and identification of metastasis-associated proteins using a subtractive immunization strategy

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik J

    2009-01-01

    and technologically challenging, and no ideal method is currently available. Here, we describe a strategy that allows scanning of the entire cell surface and identification of molecules that exhibit altered expression between two cell types. Concurrently, this method gives rise to valuable reagents...... for further characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A...... neuroendocrine tumors while exhibiting no or very weak reactivity with normal tissues. mAb 15C7 stained a variety of cancers as well as some normal lymphoid organs and was subsequently identified to react with HLA-DR-beta. A third mAb, 31D7, that also specifically recognized HLA-DR-beta was capable of inhibiting...

  16. Proteomic analysis of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of retinoblastoma-interacting-zinc-finger protein.

    Science.gov (United States)

    Chambery, Angela; Farina, Annarita; Di Maro, Antimo; Rossi, Mariangela; Abbondanza, Ciro; Moncharmont, Bruno; Malorni, Livia; Cacace, Giuseppina; Pocsfalvi, Gabriella; Malorni, Antonio; Parente, Augusto

    2006-05-01

    To identify a growth-promoting activity related to retinoblastoma-interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products. PMID:16674107

  17. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.

    Science.gov (United States)

    Connolly, Joseph P; Comerci, Diego; Alefantis, Timothy G; Walz, Alexander; Quan, Marian; Chafin, Ryan; Grewal, Paul; Mujer, Cesar V; Ugalde, Rodolfo A; DelVecchio, Vito G

    2006-07-01

    Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans. PMID:16739129

  18. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

    Science.gov (United States)

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-01-01

    A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

  19. Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde

    International Nuclear Information System (INIS)

    Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3σ and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe. - Graphical abstract: Display Omitted Research highlights: → 6-bromoisovanillin induced spindle disruption and sustained mitotic arrest, consequently resulted in mitotic catastrophe. → Proteomic profiling identified 137 differentially expressed proteins associated mitotic catastrophe. → The 14-3-3-mediated signaling network was the most significantly enriched for the altered proteins. → The macromolecule complex assembly, cell cycle, chromatin remodeling and DNA repair, tubulin organization were also shown involved in mitotic catastrophe.

  20. Comparative proteomics reveals highly and differentially expressed proteins in field-collected and laboratory-cultured blooming cells of the diatom Skeletonema costatum.

    Science.gov (United States)

    Zhang, Hao; Wang, Da-Zhi; Xie, Zhang-Xian; Zhang, Shu-Fei; Wang, Ming-Hua; Lin, Lin

    2015-10-01

    Diatoms are a major phytoplankton group causing extensive blooms in the ocean. However, little is known about the intracellular biological processes occurring during the blooming period. This study compared the protein profiles of field-collected and laboratory-cultured blooming cells of Skeletonema costatum, and identified highly and differentially expressed proteins using the shotgun proteomic approach. A total of 1372 proteins were confidently identified with two or more peptides. Among them, 222 and 311 proteins were unique to the laboratory and field samples respectively. Proteins involved in photosynthesis, translation, nucleosome assembly, carbohydrate and energy metabolism dominated the protein profiles in both samples. However, different features of specific proteins were also found: proteins participated in light harvesting, photosynthetic pigment biosynthesis, photoprotection, cell division and redox homeostasis were highly detected in the field sample, whereas proteins involved in translation, amino acid and protein metabolic processes, and nitrogen and carbon assimilation presented high detection rates in the laboratory sample. ATP synthase cf1 subunit beta and light harvest complex protein were the most abundant protein in the laboratory and field samples respectively. These results indicated that S. costatum had evolved adaptive mechanisms to the changing environment, and integrating field and laboratory proteomic data should provide comprehensive understanding of bloom mechanisms. PMID:26014042

  1. Synthetic protease inhibitor-induced inclusions in PC12 cells Potential proteomic characterization of six subunits in the 26S proteasome

    Institute of Scientific and Technical Information of China (English)

    Mingxiu Tian; Xing'an Li; Yingjiu Zhang; Yihong Hu; Ming Chang; Tao Liu; Danping Wang; Yu Zhang; Linsen Hu

    2010-01-01

    Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic proteasome inhibitor-induced inclusions in PC12 cells contained six subunits in the 26S proteasome. In the present study, mass spectrometry analysis of single protein spots resolved by two-dimensional gel electrophoresis and identified by bioinformatic analysis of peptide mass fingerprint (PMF) data were performed to comprehensively characterize the proteomic profile of the proteasome subunits. Results showed that six subunits in the 26S proteasome were characterized through accurate assignment by PMF data-specific protein identification in protein databases. Additionally, identification of one of the proteasome subunits was further confirmed using a subunit-specific antibody against non-adenosine triphosphatase subunit 11 of the 19S regulatory particle. Results suggest that the potential proteomic profile of six subunits in the 26S proteasome could be established from proteasome inhibitor-induced inclusions in PC12 cells.

  2. Comprehensive Proteomic Study of the Antiproliferative Activity of a Polyphenol-Enriched Rosemary Extract on Colon Cancer Cells Using Nanoliquid Chromatography-Orbitrap MS/MS.

    Science.gov (United States)

    Valdés, Alberto; Artemenko, Konstantin A; Bergquist, Jonas; García-Cañas, Virginia; Cifuentes, Alejandro

    2016-06-01

    In this work, a proteomics strategy based on nanoliquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) using an Orbitrap high-resolution mass spectrometer together with stable isotope dimethyl labeling (DML) is applied to quantitatively examine relative changes in the protein fraction of HT-29 human colon cancer cells treated with different concentrations of a polyphenol-enriched rosemary extract over the time. The major objective of this study was to gain insights into the antiproliferative mechanisms induced by rosemary polyphenols. Using this methodology, 1909 and 698 proteins were identified and quantified in cell extracts. The polyphenol-enriched rosemary extract treatment changed the expression of several proteins in a time- and concentration-dependent manner. Most of the altered proteins are implicated in the activation of Nrf2 transcription factor and the unfolded protein response. In conclusion, rosemary polyphenols induced proteomic changes that were related to the attenuation of aggresome formation and activation of autophagy to alleviate cellular stress. PMID:27103343

  3. An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses

    International Nuclear Information System (INIS)

    This work combined transcriptomics, proteomics, and microRNA (miRNA) analyses to elucidate the mechanism of natural hydroxyapatite (NHA)-induced osteogenic differentiation of mesenchymal stem cells (MSCs). First, NHA powder was obtained from pig bones and fabricated into disc-shaped samples. Subsequently, the proliferation and osteogenic differentiation of MSCs cultured on NHA were investigated. Then, proteomics was employed to detect the protein expression profiles of MSCs cultured on NHA, and the effect of NHA on MSCs was analyzed through an integrated pathway analysis (including proteomics and previous transcriptomics data) in which specific NHA-induced differentiation pathways were analyzed. The pathway nodes with expression data at both the mRNA and protein levels (mRNA–protein pairs) were filtered in differentiation-related pathways. miRNAs corresponding to these target mRNA–protein pairs were predicted, screened and tested, and the regulatory effects of miRNAs on mRNA–protein pairs were analyzed. Finally, the NHA-induced osteogenic pathways were verified. The results of an MTT assay and alkaline phosphatase (ALP) staining showed that the cell proliferation rate decreased and the osteogenic performance improved in the presence of NHA. By integrating transcriptomics and proteomics, the genes and proteins involved in 89 pathways were shown to be differentially expressed. Among them, 5 differentiation-associated pathways, in which 9 miRNAs and 8 regulated-target mRNA–protein zby inhibiting the target mRNA–protein pair HSPA8 in the MAPK signaling pathway, and miR-26a and miR-26b might inhibit adipogenic differentiation by repressing the target mRNA–protein pair HMGA1 in the adipogenesis pathway. A verification experiment for the osteogenic pathway indicated that the ERK1/2 or JNK MAPK pathways might play an important role in NHA-induced osteogenic differentiation. In conclusion, NHA affected MSCs at both the transcriptional and translational levels

  4. Quantitative iTRAQ LC-MS/MS Proteomics Reveals the Proteome Profiles of DF-1 Cells after Infection with Subgroup J Avian Leukosis Virus

    OpenAIRE

    Xiaofei Li; Qi Wang; Yanni Gao; Xiaole Qi; Yongqiang Wang; Honglei Gao; Yulong Gao; Xiaomei Wang

    2015-01-01

    Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that can induce various clinical tumors and has caused severe economic losses in China. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of ALV-J infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in DF-1 cells infected and...

  5. Integration of genomic and proteomic data to identify candidate genes in HT-29 cells after incubation with Bifidobacterium bifidum ATCC 29521.

    Science.gov (United States)

    Wang, Bao-Gui; Wu, Yaoping; Qiu, Liang; Shah, Nagendra P; Xu, Feng; Wei, Hua

    2016-09-01

    As the predominant group inhabiting the human gastrointestinal tract, bifidobacteria play a vital role in human nutrition, therapeutics, and health by shaping and maintaining the gut ecosystem, reducing blood cholesterol, and promoting the supply of nutrients. The interaction between bacterial cells and human intestinal epithelial cell lines has been studied for decades in an attempt to understand the mechanisms of action. These studies, however, have been limited by lack of genomic and proteomic database to aid in achieving comprehensive understanding of these mechanisms at molecular levels. Microarray data (GSE: 74119) coupled with isobaric tags for relative and absolute quantitation (iTRAQ) were performed to detect differentially expressed genes and proteins in HT-29 cells after incubation with Bifidobacterium bifidum. Real-time quantitative PCR, gene ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were further conducted for mRNA validation, functional annotation, and pathway identification, respectively. According to the results of microarray, 1,717 differentially expressed genes, including 1,693 upregulated and 24 downregulated genes, were selected and classified by the gene ontology database. The iTRAQ analysis identified 43 differentially expressed proteins, where 29 proteins were upregulated and 14 proteins were downregulated. Eighty-two candidate genes showing consistent differences with microarray and iTRAQ were further validated in HT-29 and Caco-2 cells by real-time quantitative PCR. Nine of the top genes showing interesting results with high confidence were further investigated in vivo in mice intestine samples. Integration of genomic and proteomic data provides an approach to identify candidate genes that are more likely to function in ubiquitin-mediated proteolysis, positive regulation of apoptosis, membrane proteins, and transferase catalysis. These findings might contribute to our understanding of molecular mechanisms regulating the

  6. Comprehensive quantitative comparison of the membrane proteome and PTM-ome of human embryonic stem cells and neural stem cells

    DEFF Research Database (Denmark)

    Braga, Marcella Nunes de Melo; Schulz, Melanie; Jakobsen, Lene;

    Introduction: Human embryonic stem cells (hESCs) can differentiate into all three germ layers and self-renew. Due to its ability to differentiate in vitro into human neural stem cells (hNSCs), which can further be differentiated into motor neurons and dopaminergic neurons, these cells are potential...... source for treatment of neurological diseases such as Parkinson´s disease. Membrane proteins are very important in cellular signaling and they are regulated by post-translational modifications such as phosphorylation and glycosylation. In order to obtain more information about important membrane proteins...... identified phosphorylated and SA glycosylated proteins, respectively. This study allowed us to identify several significantly regulated proteins during the differentiation process, including proteins involved in the early embryonic development as well as in the neural development. In the latter group of...

  7. Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool

    OpenAIRE

    Lanigan, Peter M.P.; Chan, Karen; Ninkovic, Tanya; Templer, Richard H.; French, P. M. W.; de Mello, A.J.; Willison, K R; Parker, P.J.; Neil, M. A. A.; Ces, Oscar; Klug, D. R.

    2008-01-01

    We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5–5 μm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane...

  8. Cell Transformation and Proteome Alteration in QSG7701 Cells Transfected with Hepatitis C Virus Non-structural Protein 3

    Institute of Scientific and Technical Information of China (English)

    Qiongqiong HE; Deyun FENG; Ruixue CHENG; Zhuchu CHEN; Xuxian XIAO; Zhiqiang XIAO; Cui LI; Bo LI; Pengfei ZHANG; Hui ZHENG

    2007-01-01

    Persistent hepatitis C virus (HCV) infection can cause liver cirrhosis and hepatocellular carcinoma. Non-structural protein 3 (NS3), an important part of HCV, has been implicated in the life cycle of the virus and interacts with host cellular proteins. In this study, we investigated the effect of NS3 protein on cell tranformation and related protein alteration in human hepatocyte QSG7701 cells. The results indicated that stable expression of the NS3 protein in QSG7701 cells induced transformed characters with reduced population doubling time, anchorage-independent growth and tumor development. Fifteen differentially-expressed proteins were separated and identified using 2-D electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Western blot analysis confirmed that the increase of phospho-p44/42 and phospho-p38 proteins was associated with transformed cells. These results supported the view that HCV NS3 protein plays a transforming role and provided some clues to elucidate the carcinogenesis mechanism of HCV-related hepatocellular carcinoma.

  9. Proteomics-based Identification of Proteins with Altered Expression Induced by 12-O-Tetradecanoylphorbol 13-acetate in Nasopharyngeal Carcinoma CNE2 Cells

    Institute of Scientific and Technical Information of China (English)

    Pei-Zhou JIANG; Ming GAN; Hua HUANG; Xin-Ming SHEN; Shuang WANG; Kai-Tai YAO

    2005-01-01

    Nasopharyngeal carcinoma (NPC) is a malignancy with high incidence in Southern China and South-East Asia. Etiology studies indicate that chemical carcinogen promoters, such as 12-Otetradecanoylphorbol-13-acetate (TPA), are important factors causing NPC development. However, the mechanism of the TPA effect on NPC remains unclear. In the present study, cells from a poorly differentiated squamous cell carcinoma NPC cell line, CNE2, were stimulated by TPA and proteomics technology was carried out to find protein discrepancies between control and TPA-treated cells. Results revealed that TPA treatment in CNE2 cells could upregulate the expression of "triosephosphate isomerase" and "14-3-3 protein sigma" and downregulate the expression of "reticulocalbin 1 precursor", "nucleophosmin", "mitochondrial matrix protein pl precursor", and "stathmin". The changes in the expression of these genes suggested that TPA induced CNE2 cells to antiproliferation and to apoptosis, which was confirmed by subsequent apoptosis detection. Therefore, the effects of TPA on nasopharyngeal carcinoma cells were distinct from the effects on primary epithelial cells and we suggest reasons for these differences.

  10. Quantitative iTRAQ LC-MS/MS proteomics reveals the proteome profiles of DF-1 cells after infection with subgroup J Avian leukosis virus.

    Science.gov (United States)

    Li, Xiaofei; Wang, Qi; Gao, Yanni; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2015-01-01

    Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that can induce various clinical tumors and has caused severe economic losses in China. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of ALV-J infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in DF-1 cells infected and mock-infected with ALV-J. A total of 75 cellular proteins were significantly changed, including 33 upregulated proteins and 42 downregulated proteins. The reliability of iTRAQ-LC MS/MS was confirmed via real-time PCR. Most of these proteins were related to the physiological functions of metabolic processes, biosynthetic processes, responses to stimuli, protein binding, signal transduction, cell cytoskeleton, and so forth. We also found some proteins that play important roles in apoptosis and oncogenicity. The differentially expressed proteins identified may provide valuable information to elucidate the pathogenesis of virus infection and virus-host interactions. PMID:25632391

  11. Urine in clinical proteomics.

    OpenAIRE

    Decramer, Stéphane; Gonzalez de Peredo, Anne; Breuil, Benjamin; Mischak, Harald; Monsarrat, Bernard; Bascands, Jean-Loup; Schanstra, Joost P

    2008-01-01

    Urine has become one of the most attractive biofluids in clinical proteomics as it can be obtained non-invasively in large quantities and is stable compared with other biofluids. The urinary proteome has been studied by almost any proteomics technology, but mass spectrometry-based urinary protein and peptide profiling has emerged as most suitable for clinical application. After a period of descriptive urinary proteomics the field is moving out of the discovery phase into an era of validation ...

  12. Mining the granule proteome

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Goetze, Jens P; Johnsen, Anders H

    2015-01-01

    Proteomics of secretory granules is an emerging strategy for identifying secreted proteins, including potentially novel candidate biomarkers and peptide hormones. In addition, proteomics can provide information about the abundance, localization and structure (post-translational modification) of...... granule proteins and peptides. Analytical strategies within this research line include so-called 'subtractive proteomics', 'peptidomics' and granule purification by the use of multiple gradient centrifugations. Here we review the literature, and describe the challenges and opportunities in proteomics of...

  13. Proteomics and the dynamic plasma membrane

    DEFF Research Database (Denmark)

    Sprenger, Richard R; Jensen, Ole Nørregaard

    2010-01-01

    plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma...... the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies...... required for detailed functional and comparative analysis of the dynamic plasma membrane proteome....

  14. Proteomic analysis of the effects of aged garlic extract and its FruArg component on lipopolysaccharide-induced neuroinflammatory response in microglial cells.

    Directory of Open Access Journals (Sweden)

    Hui Zhou

    Full Text Available Aged garlic extract (AGE is widely used as a dietary supplement, and is claimed to promote human health through anti-oxidant/anti-inflammatory activities with hypolipidemic, antiplatelet and neuroprotective effects. Prior studies of AGE have mainly focused on its organosulfur compounds, with little attention paid to its carbohydrate derivatives, such as N-α-(1-deoxy-D-fructos-1-yl-L-arginine (FruArg. The goal of this study is to investigate actions of AGE and FruArg on antioxidative and neuroinflammatory responses in lipopolysaccharide (LPS-activated murine BV-2 microglial cells using a proteomic approach. Our data show that both AGE and FruArg can significantly inhibit LPS-induced nitric oxide (NO production in BV-2 cells. Quantitative proteomic analysis by combining two dimensional differential in-gel electrophoresis (2D-DIGE with mass spectrometry revealed that expressions of 26 proteins were significantly altered upon LPS exposure, while levels of 20 and 21 proteins exhibited significant changes in response to AGE and FruArg treatments, respectively, in LPS-stimulated BV-2 cells. Notably, approximate 78% of the proteins responding to AGE and FruArg treatments are in common, suggesting that FruArg is a major active component of AGE. MULTICOM-PDCN and Ingenuity Pathway Analyses indicate that the proteins differentially affected by treatment with AGE and FruArg are involved in inflammatory responses and the Nrf2-mediated oxidative stress response. Collectively, these results suggest that AGE and FruArg attenuate neuroinflammatory responses and promote resilience in LPS-activated BV-2 cells by suppressing NO production and by regulating expression of multiple protein targets associated with oxidative stress.

  15. A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line

    Directory of Open Access Journals (Sweden)

    Corrêa Stephany

    2012-03-01

    Full Text Available Abstract Background Although chronic myeloid leukemia (CML treatment has improved since the introduction of imatinib mesylate (IM, cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR cells. Results The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC protein, minichromosome maintenance complex component 7 (MCM7 and ATP-binding cassette sub-family B (MDR/TAP member 1 (ABCB1 were capable of defining samples from CML patients as responsive or resistant to therapy. Conclusions Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.

  16. Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches

    Directory of Open Access Journals (Sweden)

    Rocchi Mara S

    2011-08-01

    Full Text Available Abstract Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI responses involving CD4+ve, CD8+ve, γ/δ TCR+ve T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4+ve T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4+ve T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4+ve cells and the subsequent identification of the stimulating components using tandem mass spectrometry.

  17. Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches.

    Science.gov (United States)

    Rocchi, Mara S; Bartley, Paul M; Inglis, Neil F; Collantes-Fernandez, Esther; Entrican, Gary; Katzer, Frank; Innes, Elisabeth A

    2011-01-01

    Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI) responses involving CD4(+ve), CD8(+ve), γ/δ TCR(+ve) T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4(+ve) T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA) were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4(+ve) T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry) was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4(+ve) cells) and the subsequent identification of the stimulating components using tandem mass spectrometry. PMID:21813001

  18. Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC approach

    Directory of Open Access Journals (Sweden)

    Pan ST

    2015-02-01

    Full Text Available Shu-Ting Pan,1,* Zhi-Wei Zhou,2,3,* Zhi-Xu He,3 Xueji Zhang,4 Tianxin Yang,5 Yin-Xue Yang,6 Dong Wang,7 Jia-Xuan Qiu,1 Shu-Feng Zhou2 1Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 4Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 5Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 7Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China *These two authors contributed equally to this work Abstract: 5,6-Dimethylxanthenone 4-acetic acid (DMXAA, also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC approach. The proteomic data showed that treatment with DMXAA

  19. Proteomics of human mitochondria

    DEFF Research Database (Denmark)

    Palmfeldt, Johan; Bross, Peter

    2016-01-01

    Proteomics have passed through a tremendous development in the recent years by the development of ever more sensitive, fast and precise mass spectrometry methods. The dramatically increased research in the biology of mitochondria and their prominent involvement in all kinds of diseases and ageing...... sensitivity of mass spectrometry technology aids in lowering this hurdle and new approaches like generation of induced pluripotent cells from somatic cells allow to produce patient-specific cellular disease models with great potential. We describe which human sample types are accessible, review the status of...... the catalog of human mitochondrial proteins and discuss proteins with dual localization in mitochondria and other cellular compartments. We describe the status and developments of pertinent mass spectrometric strategies, and the use of databases and bioinformatics. Using selected illustrative examples...

  20. Preexisting CD4+ T-cell immunity in human population to avian influenza H7N9 virus: whole proteome-wide immunoinformatics analyses.

    Directory of Open Access Journals (Sweden)

    Venkata R Duvvuri

    Full Text Available In 2013, a novel avian influenza H7N9 virus was identified in human in China. The antigenically distinct H7N9 surface glycoproteins raised concerns about lack of cross-protective neutralizing antibodies. Epitope-specific preexisting T-cell immunity was one of the protective mechanisms in pandemic 2009 H1N1 even in the absence of cross-protective antibodies. Hence, the assessment of preexisting CD4+ T-cell immunity to conserved epitopes shared between H7N9 and human influenza A viruses (IAV is critical. A comparative whole proteome-wide immunoinformatics analysis was performed to predict the CD4+ T-cell epitopes that are commonly conserved within the proteome of H7N9 in reference to IAV subtypes (H1N1, H2N2, and H3N2. The CD4+ T-cell epitopes that are commonly conserved (∼ 556 were further screened against the Immune Epitope Database (IEDB to validate their immunogenic potential. This analysis revealed that 45.5% (253 of 556 epitopes are experimentally proven to induce CD4+ T-cell memory responses. In addition, we also found that 23.3% of CD4+ T-cell epitopes have ≥ 90% of sequence homology with experimentally defined CD8+ T-cell epitopes. We also conducted the population coverage analysis across different ethnicities using commonly conserved CD4+ T-cell epitopes and corresponding HLA-DRB1 alleles. Interestingly, the indigenous populations from Canada, United States, Mexico and Australia exhibited low coverage (28.65% to 45.62% when compared with other ethnicities (57.77% to 94.84%. In summary, the present analysis demonstrate an evidence on the likely presence of preexisting T-cell immunity in human population and also shed light to understand the potential risk of H7N9 virus among indigenous populations, given their high susceptibility during previous pandemic influenza events. This information is crucial for public health policy, in targeting priority groups for immunization programs.

  1. Preliminary Proteomic Analysis of A549 Cells Infected with Avian Influenza Virus H7N9 and Influenza A Virus H1N1

    Science.gov (United States)

    Ding, Xiaoman; Lu, Jiahai; Yu, Ruoxi; Wang, Xin; Wang, Ting; Dong, Fangyuan; Peng, Bo; Wu, Weihua; Liu, Hui; Geng, Yijie; Zhang, Renli; Ma, Hanwu; Cheng, Jinquan; Yu, Muhua; Fang, Shisong

    2016-01-01

    A newly emerged H7N9 influenza virus poses high risk to human beings. However, the pathogenic mechanism of the virus remains unclear. The temporal response of primary human alveolar adenocarcinoma epithelial cells (A549) infected with H7N9 influenza virus and H1N1 influenza A virus (H1N1, pdm09) were evaluated using the proteomics approaches (2D-DIGE combined with MALDI-TOF-MS/MS) at 24, 48 and 72 hours post of the infection (hpi). There were 11, 12 and 33 proteins with significant different expressions (P<0.05) at 24, 48 and 72hpi, especially F-actin-capping protein subunit alpha-1 (CAPZA1), Ornithine aminotransferase (OAT), Poly(rC)-binding protein 1 (PCBP1), Eukaryotic translation initiation factor 5A-1 (EIF5A) and Platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2) were validated by western-blot analysis. The functional analysis revealed that the differential proteins in A549 cells involved in regulating cytopathic effect. Among them, the down-regulation of CAPZA1, OAT, PCBP1, EIF5A are related to the death of cells infected by H7N9 influenza virus. This is the first time show that the down-regulation of PAFAH1B2 is related to the later clinical symptoms of patients infected by H7N9 influenza virus. These findings may improve our understanding of pathogenic mechanism of H7N9 influenza virus in proteomics. PMID:27223893

  2. Integrated transcriptomic and proteomic analysis identifies protein kinase CK2 as a key signaling node in an inflammatory cytokine network in ovarian cancer cells

    Science.gov (United States)

    Kulbe, Hagen; Iorio, Francesco; Chakravarty, Probir; Milagre, Carla S.; Moore, Robert; Thompson, Richard G.; Everitt, Gemma; Canosa, Monica; Montoya, Alexander; Drygin, Denis; Braicu, Ioana; Sehouli, Jalid; Saez-Rodriguez, Julio; Cutillas, Pedro R.; Balkwill, Frances R.

    2016-01-01

    We previously showed how key pathways in cancer-related inflammation and Notch signaling are part of an autocrine malignant cell network in ovarian cancer. This network, which we named the “TNF network”, has paracrine actions within the tumor microenvironment, influencing angiogenesis and the immune cell infiltrate. The aim of this study was to identify critical regulators in the signaling pathways of the TNF network in ovarian cancer cells that might be therapeutic targets. To achieve our aim, we used a systems biology approach, combining data from phospho-proteomic mass spectrometry and gene expression array analysis. Among the potential therapeutic kinase targets identified was the protein kinase Casein kinase II (CK2). Knockdown of CK2 expression in malignant cells by siRNA or treatment with the specific CK2 inhibitor CX-4945 significantly decreased Notch signaling and reduced constitutive cytokine release in ovarian cancer cell lines that expressed the TNF network as well as malignant cells isolated from high grade serous ovarian cancer ascites. The expression of the same cytokines was also inhibited after treatment with CX-4945 in a 3D organotypic model. CK2 inhibition was associated with concomitant inhibition of proliferative activity, reduced angiogenesis and experimental peritoneal ovarian tumor growth. In conclusion, we have identified kinases, particularly CK2, associated with the TNF network that may play a central role in sustaining the cytokine network and/or mediating its effects in ovarian cancer. PMID:26871292

  3. Proteogenomics Dashboard for the Human Proteome Project.

    Science.gov (United States)

    Tabas-Madrid, Daniel; Alves-Cruzeiro, Joao; Segura, Victor; Guruceaga, Elizabeth; Vialas, Vital; Prieto, Gorka; García, Carlos; Corrales, Fernando J; Albar, Juan Pablo; Pascual-Montano, Alberto

    2015-09-01

    dasHPPboard is a novel proteomics-based dashboard that collects and reports the experiments produced by the Spanish Human Proteome Project consortium (SpHPP) and aims to help HPP to map the entire human proteome. We have followed the strategy of analog genomics projects like the Encyclopedia of DNA Elements (ENCODE), which provides a vast amount of data on human cell lines experiments. The dashboard includes results of shotgun and selected reaction monitoring proteomics experiments, post-translational modifications information, as well as proteogenomics studies. We have also processed the transcriptomics data from the ENCODE and Human Body Map (HBM) projects for the identification of specific gene expression patterns in different cell lines and tissues, taking special interest in those genes having little proteomic evidence available (missing proteins). Peptide databases have been built using single nucleotide variants and novel junctions derived from RNA-Seq data that can be used in search engines for sample-specific protein identifications on the same cell lines or tissues. The dasHPPboard has been designed as a tool that can be used to share and visualize a combination of proteomic and transcriptomic data, providing at the same time easy access to resources for proteogenomics analyses. The dasHPPboard can be freely accessed at: http://sphppdashboard.cnb.csic.es. PMID:26144527

  4. Induced pluripotent stem cells: origins, applications, and future perspectives*

    OpenAIRE

    Zhao, Jing; Jiang, Wen-jie; Sun, Chen; Hou, Cong-zhe; Yang, Xiao-Mei; Gao, Jian-gang

    2013-01-01

    Embryonic stem (ES) cells are widely used for different purposes, including gene targeting, cell therapy, tissue repair, organ regeneration, and so on. However, studies and applications of ES cells are hindered by ethical issues regarding cell sources. To circumvent ethical disputes, great efforts have been taken to generate ES cell-like cells, which are not derived from the inner cell mass of blastocyst-stage embryos. In 2006, Yamanaka et al. first reprogrammed mouse embryonic fibroblasts in...

  5. Investigating evolutionary perspective of carcinogenesis with single-cell transcriptome analysis

    Institute of Scientific and Technical Information of China (English)

    Xi Zhang; Cheng Zhang; Zhongjun Li; Jiangjian Zhong; Leslie P. Weiner; Jiang F. Zhong

    2013-01-01

    We developed phase-switch microfluidic devices for molecular profiling of a large number of single cells. Whole genome microarrays and RNA-sequencing are commonly used to determine the expression levels of genes in cell lysates (a physical mix of millions of cells) for inferring gene functions. However, cellular heterogeneity becomes an inherent noise in the measurement of gene expression. The unique molecular characteristics of individual cells, as well as the temporal and quantitative information of gene expression in cells, are lost when averaged among all cells in cell lysates. Our single-cell technology overcomes this limitation and enables us to obtain a large number of single-cell transcriptomes from a population of cells. A collection of single-cell molecular profiles allows us to study carcinogenesis from an evolutionary perspective by treating cancer as a diverse population of cells with abnormal molecular characteristics. Because a cancer cellpopulation contains cells at various stages of development toward drug resistance, clustering similar single-cell molecular profiles could reveal how drug-resistant sub-clones evolve during cancer treatment. Here, we discuss how single-celltranscriptome analysis technology could enable the study of carcinogenesis from an evolutionary perspective and the development of drug-resistance in leukemia. The single-cell transcriptome analysis reported here could have a direct and significant impact on current cancer treatments and future personalized cancer therapies.

  6. Proteomic Investigation of Rhizoctonia solani AG 4 Identifies Secretome and Mycelial Proteins with Roles in Plant Cell Wall Degradation and Virulence.

    Science.gov (United States)

    Lakshman, Dilip K; Roberts, Daniel P; Garrett, Wesley M; Natarajan, Savithiry S; Darwish, Omar; Alkharouf, Nadim; Pain, Arnab; Khan, Farooq; Jambhulkar, Prashant P; Mitra, Amitava

    2016-04-20

    Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. This study used a proteomics approach to characterize both intracellular proteins and the secretome of R. solani AG 4 isolate Rs23A under several growth conditions, the secretome being highly important in pathogenesis. From over 500 total secretome and soluble intracellular protein spots from 2-D gels, 457 protein spots were analyzed and 318 proteins positively matched with fungal proteins of known function by comparison with available R. solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups and for some proteins their putative roles in plant cell wall degradation and virulence. The majority of the secreted proteins were grouped to extracellular regions and contain hydrolase activity. PMID:27019116

  7. Proteomic Investigation of Rhizoctonia solani AG 4 Identifies Secretome and Mycelial Proteins with roles in Plant Cell Wall Degradation and Virulence

    KAUST Repository

    Lakshman, Dilip

    2016-03-28

    Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. Here we used a proteomics approach to characterize both intracellular proteins and the secretome of R. solani AG 4 isolate Rs23A under several growth conditions; the secretome being highly important in pathogenesis. From over 500 total secretome and soluble intracellular protein spots from 2-D gels, 457 protein spots were analyzed and 318 proteins positively matched with fungal proteins of known function by comparison with available R. solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups; and for some proteins their putative roles in plant cell wall degradation and virulence. The majority of the secreted proteins were grouped to extracellular regions and contain hydrolase activity.

  8. Effects of Elaidic Acid on Lipid Metabolism in HepG2 Cells, Investigated by an Integrated Approach of Lipidomics, Transcriptomics and Proteomics

    DEFF Research Database (Denmark)

    Vendel Nielsen, Lone; Hansen, Toke Peter Krogager; Young, Clifford;

    2013-01-01

    Trans fatty acid consumption in the human diet can cause adverse health effects, such as cardiovascular disease, which is associated with higher total cholesterol, a higher low density lipoprotein-cholesterol level and a decreased high density lipoprotein-cholesterol level. The aim of the study was...... to elucidate the hepatic response to the most abundant trans fatty acid in the human diet, elaidic acid, to help explain clinical findings on the relationship between trans fatty acids and cardiovascular disease. The human HepG2 cell line was used as a model to investigate the hepatic response to...... elaidic acid in a combined proteomic, transcriptomic and lipidomic approach. We found many of the proteins responsible for cholesterol synthesis up-regulated together with several proteins involved in the esterification and hepatic import/export of cholesterol. Furthermore, a profound remodeling of the...

  9. Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach

    Directory of Open Access Journals (Sweden)

    Reddivari Lavanya

    2011-08-01

    Full Text Available Abstract Background We and others have previously reported that resveratrol (RSV suppresses colon cancer cell proliferation and elevates apoptosis in vitro and/or in vivo, however molecular mechanisms are not fully elucidated. Particularly, little information is available on RSV's effects on metabolic pathways and the cell-extra cellular matrix (ECM communication that are critical for cancer cell growth. To identify important targets of RSV, we analyzed whole protein fractions from HT-29 advanced human colon cancer cell line treated with solvent control, IGF-1 (10 nM and RSV (150 μM using LC/MS/MS-Mud PIT (Multidimensional Protein Identification Technology. Results Pentose phosphate pathway (PPP, a vital metabolic pathway for cell cycle progression, was elevated and suppressed by IGF-1 and RSV, respectively in the HT-29 cell line. Enzymatic assays confirmed RSV suppression of glucose-6 phosphate dehydrogenase (rate limiting and transketolase, key enzymes of the PPP. RSV (150 μM suppressed, whereas IGF-1 (10 nM elevated focal adhesion complex (FAC proteins, talin and pFAK, critical for the cell-ECM communication. Western blotting analyses confirmed the suppression or elevation of these proteins in HT-29 cancer cells treated with RSV or IGF-1, respectively. Conclusions Proteomic analysis enabled us to establish PPP and the talin-pFAK as targets of RSV which suppress cancer cell proliferation and induce apoptosis in the colon cancer cell line HT-29. RSV (150 μM suppressed these pathways in the presence and absence of IGF-1, suggesting its role as a chemo-preventive agent even in obese condition.

  10. Analysis of the membrane proteome of ciprofloxacin-resistant macrophages by stable isotope labeling with amino acids in cell culture (SILAC.

    Directory of Open Access Journals (Sweden)

    Nancy E Caceres

    Full Text Available Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient. Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1 and 69 (F2 showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter.

  11. Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination

    OpenAIRE

    Flodrová, D. (Dana); Benkovská, D. (Dagmar); Macejová, D.; Bialesova, L.; Hunakova, L.; Brtko, J.; Bobálová, J. (Janette)

    2015-01-01

    Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a ...

  12. Role of the proteome in phytohormonal signaling.

    Science.gov (United States)

    Černý, Martin; Novák, Jan; Habánová, Hana; Cerna, Hana; Brzobohatý, Břetislav

    2016-08-01

    Phytohormones are orchestrators of plant growth and development. A lot of time and effort has been invested in attempting to comprehend their complex signaling pathways but despite success in elucidating some key components, molecular mechanisms in the transduction pathways are far from being resolved. The last decade has seen a boom in the analysis of phytohormone-responsive proteins. Abscisic acid, auxin, brassinosteroids, cytokinin, ethylene, gibberellins, nitric oxide, oxylipins, strigolactones, salicylic acid - all have been analyzed to various degrees. For this review, we collected data from proteome-wide analyses resulting in a list of over 2000 annotated proteins from Arabidopsis proteomics and nearly 500 manually filtered protein families merged from all the data available from different species. We present the currently accepted model of phytohormone signaling, highlight the contributions made by proteomic-based research and describe the key nodes in phytohormone signaling networks, as revealed by proteome analysis. These include ubiquitination and proteasome mediated degradation, calcium ion signaling, redox homeostasis, and phosphoproteome dynamics. Finally, we discuss potential pitfalls and future perspectives in the field. This article is part of a Special Issue entitled: Plant Proteomics - a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. PMID:26721743

  13. The Potato Tuber Mitochondrial Proteome

    DEFF Research Database (Denmark)

    Salvato, Fernanda; Havelund, Jesper F; Chen, Mingjie;

    2014-01-01

    Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum ‘Folva’) and their proteome investigated. Proteins...... that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy...

  14. New perspectives in human stem cell therapeutic research

    OpenAIRE

    Trounson Alan

    2009-01-01

    Abstract Human stem cells are in evaluation in clinical stem cell trials, primarily as autologous bone marrow studies, autologous and allogenic mesenchymal stem cell trials, and some allogenic neural stem cell transplantation projects. Safety and efficacy are being addressed for a number of disease state applications. There is considerable data supporting safety of bone marrow and mesenchymal stem cell transplants but the efficacy data are variable and of mixed benefit. Mechanisms of action o...

  15. Proteomic Profiling of Hematopoietic Stem/Progenitor Cells after a Whole Body Exposure of CBA/CaJ Mice to Titanium (48Ti Ions

    Directory of Open Access Journals (Sweden)

    Kanokporn Noy Rithidech

    2015-07-01

    Full Text Available Myeloid leukemia (ML is one of the major health concerns from exposure to radiation. However, the risk assessment for developing ML after exposure to space radiation remains uncertain. To reduce the uncertainty in risk prediction for ML, a much increased understanding of space radiation-induced changes in the target cells, i.e., hematopoietic stem/progenitor cells (HSPCs, is critically important. We used the label-free quantitative mass spectrometry (LFQMS proteomic approach to determine the expression of protein in HSPC-derived myeloid colonies obtained at an early time-point (one week and a late time-point (six months after an acute whole body exposure of CBA/CaJ mice to a total dose of 0, 0.1, 0.25, or 0.5 Gy of heavy-ion titanium (48Ti ions, which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48Ti ions served as non-irradiated sham controls. There were five mice per treatment groups at each harvest time. The Trans-Proteomic Pipeline (TPP was used to assign a probability of a particular protein being in the sample. A proof-of-concept based Ingenuity Pathway Analysis (IPA was used to characterize the functions, pathways, and networks of the identified proteins. Alterations of expression levels of proteins detected in samples collected at one week (wk post-irradiation reflects acute effects of exposure to 48Ti ions, while those detected in samples collected at six months (mos post-irradiation represent protein expression profiles involved in the induction of late-occurring damage (normally referred to as genomic instability. Our results obtained by using the IPA analyses indicate a wide array of signaling pathways involved in response to 1 GeV/n 48Ti ions at both harvest times. Our data also demonstrate that the patterns of protein expression profiles are dose and time dependent. The majority of proteins with altered expression levels are involved in cell cycle control, cellular growth and

  16. E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response.

    Science.gov (United States)

    Chopjitt, Peechanika; Pientong, Chamsai; Sunthamala, Nuchsupha; Kongyingyoes, Bunkerd; Haonon, Ornuma; Boonmars, Thidarut; Kikawa, Satomi; Nakahara, Tomomi; Kiyono, Tohru; Ekalaksananan, Tipaya

    2016-09-01

    HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant. PMID:27392712

  17. Proteomic analysis of novel targets associated with the enhancement of TrkA-induced SK-N-MC cancer cell death caused by NGF

    Science.gov (United States)

    Jung, Eun Joo; Chung, Ky Hyun; Bae, Dong-Won; Kim, Choong Won

    2016-01-01

    Nerve growth factor (NGF) is known to regulate both cancer cell survival and death signaling, depending on the cellular circumstances, in various cell types. In this study, we showed that NGF strongly upregulated the protein level of tropomyosin-related kinase A (TrkA) in TrkA-inducible SK-N-MC cancer cells, resulting in increases in various TrkA-dependent cellular processes, including the phosphorylation of c-Jun N-terminal kinase (JNK) and caspase-8 cleavage. In addition, NGF enhanced TrkA-induced morphological changes and cell death, and this effect was significantly suppressed by the JNK inhibitor SP600125, but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. To investigate novel targets associated with the enhancement of TrkA-induced SK-N-MC cell death caused by NGF, we performed Coomassie Brilliant Blue staining and two-dimensional (2D) proteomic analysis in TrkA-inducible SK-N-MC cells. We identified 31 protein spots that were either greatly upregulated or downregulated by TrkA during NGF treatment using matrix-associated laser desorption/ionization time of flight/time of flight mass spectrometry, and we analyzed the effects of SP600125 and wortmannin on the spots. Interestingly, 11 protein spots, including heterogeneous nuclear ribonucleoprotein K (hnRNP K), lamin B1 and TAR DNA-binding protein (TDP43), were significantly influenced by SP600125, but not by wortmannin. Moreover, the NGF/TrkA-dependent inhibition of cell viability was significantly enhanced by knockdown of hnRNP K using small interfering RNA, demonstrating that hnRNP K is a novel target associated with the regulation of TrkA-dependent SK-N-MC cancer cell death enhanced by NGF. PMID:27229480

  18. Use and Needs in Contexts : An Ethnographic Study on Cell Phone Use from a Contextual Usability Perspective

    OpenAIRE

    Friberg, Hanna

    2003-01-01

    The focus of this thesis is usability of an everyday used product; the cell phone seen from a Human – Computer Interaction perspective. The purpose with the thesis is to create an understanding of how cell phones are used by persons in natural /public settings and in everyday activities. Further the purpose is to describe users’ experiences of using cell phones. In this study, ethnography was used as method. The theoretical framework is the contextual usability perspective. The cell phone is ...

  19. PROTEOMICS in aquaculture

    DEFF Research Database (Denmark)

    Rodrigues, Pedro M.; Silva, Tomé S.; Dias, Jorge;

    2012-01-01

    proteomics in seafood biology research. Proteomics, as a powerful comparative tool, has therefore been increasingly used over the last decade to address different questions in aquaculture, regarding welfare, nutrition, health, quality, and safety. In this paper we will give an overview of these biological...... questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined...... nutritional, health or quality properties for functional foods and the integration of proteomics techniques in addressing this challenging issue. This article is part of a Special Issue entitled: Farm animal proteomics....

  20. Proteomic evaluation of MCF-7 human breast cancer cells after treatment with retinoic acid isomers: Preliminary insights

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Benkovská, Dagmar; Macejová, D.; Bialešová, L.; Brtko, J.; Bobálová, Janette

    Brno: Ústav analytické chemie AV ČR, v. v. i, 2012 - (Foret, F.; Křenková, J.; Guttman, A.; Klepárník, K.; Boček, P.), s. 146-150 ISBN 978-80-904959-1-3. [CECE 2012. International Interdisciplinary Meeting on Bioanalysis /9./. Brno (CZ), 01.11.2012-02.11.2012] R&D Projects: GA MŠk(CZ) EE2.3.20.0182; GA MŠk(CZ) 7AMB12SK151 Institutional support: RVO:68081715 Keywords : retinoids * breast cancer * proteomic analysis Subject RIV: CB - Analytical Chemistry, Separation http://hdl.handle.net/11104/0214569

  1. Micro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level.

    Science.gov (United States)

    Bensaddek, Dalila; Narayan, Vikram; Nicolas, Armel; Murillo, Alejandro Brenes; Gartner, Anton; Kenyon, Cynthia J; Lamond, Angus I

    2016-02-01

    Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-associated factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd), an open access, searchable database resource. PMID:26552604

  2. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors.

    Science.gov (United States)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels H H; Borregaard, Niels

    2013-10-01

    Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes. PMID:23650620

  3. Unraveling the pathogenesis of type 1 diabetes with proteomics: present and future directions

    DEFF Research Database (Denmark)

    Sparre, Thomas; Larsen, Martin Røssel; Heding, Peter E; Karlsen, Allan E; Jensen, Ole N; Pociot, Flemming Michael

    2005-01-01

    . Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies...

  4. Hilar mossy cells of the dentate gyrus: a historical perspective

    Directory of Open Access Journals (Sweden)

    Helen E Scharfman

    2013-01-01

    Full Text Available The circuitry of the dentate gyrus of the hippocampus is unique compared to other hippocampal subfields because there are two glutamatergic principal cells instead of one: granule cells, which are the vast majority of the cells in the dentate gyrus, and the so-called ‘mossy cells.’ The distinctive appearance of mossy cells, the extensive divergence of their axons, and their vulnerability to excitotoxicity relative to granule cells has led to a great deal of interest in mossy cells. Nevertheless, there is no consensus about the normal functions of mossy cells and the implications of their vulnerability. There even seems to be some ambiguity about exactly what mossy cells are. Here we review initial studies of mossy cells, characteristics that define them, and suggest a practical definition to allow investigators to distinguish mossy cells from other hilar neurons even if all morphological and physiological information is unavailable due to technical limitations of their experiments. In addition, hypotheses are discussed about the role of mossy cells in the dentate gyrus network, reasons for their vulnerability and their implications for disease.

  5. New perspectives in human stem cell therapeutic research

    Directory of Open Access Journals (Sweden)

    Trounson Alan

    2009-06-01

    Full Text Available Abstract Human stem cells are in evaluation in clinical stem cell trials, primarily as autologous bone marrow studies, autologous and allogenic mesenchymal stem cell trials, and some allogenic neural stem cell transplantation projects. Safety and efficacy are being addressed for a number of disease state applications. There is considerable data supporting safety of bone marrow and mesenchymal stem cell transplants but the efficacy data are variable and of mixed benefit. Mechanisms of action of many of these cells are unknown and this raises the concern of unpredictable results in the future. Nevertheless there is considerable optimism that immune suppression and anti-inflammatory properties of mesenchymal stem cells will be of benefit for many conditions such as graft versus host disease, solid organ transplants and pulmonary fibrosis. Where bone marrow and mesenchymal stem cells are being studied for heart disease, stroke and other neurodegenerative disorders, again progress is mixed and mostly without significant benefit. However, correction of multiple sclerosis, at least in the short term is encouraging. Clinical trials on the use of embryonic stem cell derivatives for spinal injury and macular degeneration are beginning and a raft of other clinical trials can be expected soon, for example, the use of neural stem cells for killing inoperable glioma and embryonic stem cells for regenerating β islet cells for diabetes. The change in attitude to embryonic stem cell research with the incoming Obama administration heralds a new co-operative environment for study and evaluation of stem cell therapies. The Californian stem cell initiative (California Institute for Regenerative Medicine has engendered global collaboration for this new medicine that will now also be supported by the US Federal Government. The active participation of governments, academia, biotechnology, pharmaceutical companies, and private investment is a powerful consortium for

  6. Proteomic screening of glucose-responsive and glucose non-reponsive MIN-6 beta cells reveals differential expression of protein involved in protein folding, secretion and oxidative stress

    DEFF Research Database (Denmark)

    Dowling, P.; O´Driscoll, L.; O´Sullivan, F.;

    2006-01-01

    The glucose-sensitive insulin-secretion (GSIS) phenotype is relatively unstable in long-term culture of beta cells. The purpose of this study was to investigate relative changes in the proteome between glucose-responsive (low passage) and glucose non-responsive (high passage) murine MIN-6...... pancreatic beta cells. The 2D-DIGE and subsequent DeCyder analysis detected 3351 protein spots in the pH range of 4-7. Comparing MIN-6(H) to MIN-6(L) and using a threshold of 1.2-fold, the number of proteins with a decrease in expression level was 152 (4.5%), similar was 3140 (93.7%) and increased 59 (1...... protein 29 (ERp29); 78-kDa glucose-related protein, (GRP78); 94-kDa glucose-related protein (GRP94); protein disulphide isomerase; carbonyl reductase 3; peroxidoxin 4 and superoxide dismutase 1. These results suggest that non-GSIS MIN-6 cells do not have the same ability/capacity of glucose-responsive MIN...

  7. Comparative proteomics of uropathogenic Escherichia coli during growth in human urine identify UCA-like (UCL) fimbriae as an adherence factor involved in biofilm formation and binding to uroepithelial cells.

    Science.gov (United States)

    Wurpel, Daniël J; Totsika, Makrina; Allsopp, Luke P; Webb, Richard I; Moriel, Danilo G; Schembri, Mark A

    2016-01-10

    Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infection (UTI) in humans. For the successful colonisation of the human urinary tract, UPEC employ a diverse collection of secreted or surface-exposed virulence factors including toxins, iron acquisition systems and adhesins. In this study, a comparative proteomic approach was utilised to define the UPEC pan and core surface proteome following growth in pooled human urine. Identified proteins were investigated for subcellular origin, prevalence and homology to characterised virulence factors. Fourteen core surface proteins were identified, as well as eleven iron uptake receptor proteins and four distinct fimbrial types, including type 1, P, F1C/S and a previously uncharacterised fimbrial type, designated UCA-like (UCL) fimbriae in this study. These pathogenicity island (PAI)-associated fimbriae are related to UCA fimbriae of Proteus mirabilis, associated with UPEC and exclusively found in members of the E. coli B2 and D phylogroup. We further demonstrated that UCL fimbriae promote significant biofilm formation on abiotic surfaces and mediate specific attachment to exfoliated human uroepithelial cells. Combined, this study has defined the surface proteomic profiles and core surface proteome of UPEC during growth in human urine and identified a new type of fimbriae that may contribute to UTI. PMID:26546558

  8. Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation

    DEFF Research Database (Denmark)

    Emdal, Kristina B; Pedersen, Anna-Kathrine; Bekker-Jensen, Dorte B; Tsafou, Kalliopi P; Horn, Heiko; Lindner, Sven; Schulte, Johannes H; Eggert, Angelika; Jensen, Lars Juhl; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then...

  9. Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE.

    Science.gov (United States)

    Kalayou, Shewit; Granum, Cesilie; Berntsen, Hanne Friis; Groseth, Per Kristian; Verhaegen, Steven; Connolly, Lisa; Brandt, Ingvar; de Souza, Gustavo Antonio; Ropstad, Erik

    2016-03-30

    Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions

  10. Plant nuclear proteomics for unraveling physiological function.

    Science.gov (United States)

    Yin, Xiaojian; Komatsu, Setsuko

    2016-09-25

    The nucleus is the subcellular organelle that functions as the regulatory hub of the cell and is responsible for regulating several critical cellular functions, including cell proliferation, gene expression, and cell survival. Nuclear proteomics is a useful approach for investigating the mechanisms underlying plant responses to abiotic stresses, including protein-protein interactions, enzyme activities, and post-translational modifications. Among abiotic stresses, flooding is a major limiting factor for plant growth and yields, particularly for soybean. In this review, plant nuclei purification methods, modifications of plant nuclear proteins, and recent contributions to the field of plant nuclear proteomics are summarized. In addition, to reveal the upstream regulating mechanisms controlling soybean responses to flooding stress, the functions of flooding-responsive nuclear proteins are reviewed based on the results of nuclear proteomic analysis of soybean in the early stages of flooding stress. PMID:27004615

  11. Approaching clinical proteomics: current state and future fields of application in fluid proteomics

    OpenAIRE

    Apweiler, Rolf; Aslanidis, Charalampos; Deufel, Thomas; Gerstner, Andreas; Hansen, Jens; Hochstrasser, Dennis; Kellner, Roland; Kubicek, Markus; Lottspeich, Friedrich; Maser, Edmund; Mewes, Hans-Werner; Meyer, Helmut E.; Müllner, Stefan; Mutter, Wolfgang; Neumaier, Michael

    2009-01-01

    The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics. The nature of the human body fluid proteome with its large dynamic range of pr...

  12. Comparative proteomic analysis implicates eEF2 as a novel target of PI3Kγ in the MDA-MB-231 metastatic breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Niu Meizhi

    2013-01-01

    Full Text Available Abstract Background Cancer cell migration is fundamentally required for breast tumour invasion and metastasis. The insulin-like growth factor 1 tyrosine kinase receptor (IGF-1R and the chemokine G-protein coupled receptor, CXCR4 have been shown to play an important role in breast cancer metastasis. Our previous study has shown that IGF-1R can transactivate CXCR4 via a physical association in the human MDA-MB-231 metastatic breast cancer cell line and that this plays a key role in IGF-I-induced migration of these cells. In the present study we used pharmacological inhibition and RNAi to identify PI3Kγ as an important migration signalling molecule downstream of receptor transactivation in MDA-MB-231 cells. To identify PI3Kγ-regulated proteins upon transactivation of CXCR4 by IGF-I, we undertook a comparative proteomics approach using 2-D- Fluorescence Difference Gel Electrophoresis (DIGE and identified the proteins by mass spectrometry. Results These experiments identified eukaryotic elongation factor 2 (eEF2 as a novel downstream target of PI3Kγ after activation of the IGF-1R-CXCR4 heterodimer by IGF-I. Further analysis demonstrated that eEF2 is phosphorylated in MDA-MB-231 cells in response to IGF-I and that this is dependent on PI3Kγ activity. Conclusions Our data imply a novel role for PI3Kγ in facilitating cell migration by regulating phosphorylation of eEF2.

  13. Cell therapy and wound healing - state of art and perspectives

    Czech Academy of Sciences Publication Activity Database

    Smetana, Karel; Dvořánková, B.; Labský, Jiří; Holíková, Z.

    Daejon : Pai Chai University, 2002 - (Kim, H.; Kim, T.; Park, J.), s. 77-80 [International Symposium on Advanced Materials /4./. Daejon (KR), 03.06.2002-07.06.2002] R&D Projects: GA MŠk LN00A065; GA ČR GA203/00/1310; GA AV ČR IBS4050005 Institutional research plan: CEZ:AV0Z4050913 Keywords : epidermal stem cell * cell therapy * wound healing Subject RIV: EA - Cell Biology

  14. The Succinated Proteome

    Energy Technology Data Exchange (ETDEWEB)

    Merkley, Eric D.; Metz, Thomas O.; Smith, Richard D.; Baynes, John; Frizell, Norma

    2014-03-30

    Succination is a chemical modification of cysteine in protein by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes. Increased succination of proteins is also detected in the kidney of a fumarase conditional knock-out mouse which develops renal tumors. Keap1, the gatekeeper of the antioxidant response, was identified as a major succinated protein in renal cancer cells, suggesting that succination may play a role in activation of the antioxidant response. A wide range of proteins is subject to succination, including enzymes, adipokines, cytoskeletal proteins and ER chaperones with functional cysteine residues. There is also significant overlap between succinated and glutathionylated proteins, and with proteins containing cysteine residues that are readily oxidized to the sulfenic (cysteic) acid. Succination of adipocyte proteins is inhibited by uncouplers, which discharge the mitochondrial membrane potential (Δψm) and by ER stress inhibitors. 2SC serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic post-translational modification of proteins by proteomics approaches.

  15. Expansion of hematopoietic stem cells for transplantation: current perspectives

    Directory of Open Access Journals (Sweden)

    Schuster Jessica A

    2012-05-01

    Full Text Available Abstract Hematopoietic stem cells (HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The expansion of HSCs has remained an important goal to develop advanced cell therapies for bone marrow transplantation and many blood disorders. Over the last several decades, there have been numerous attempts to expand HSCs in vitro using purified growth factors that are known to regulate HSCs. However, these attempts have been met with limited success for clinical applications. New developments in the HSC expansion field coupled with gene therapy and stem cell transplant should encourage progression in attractive treatment options for many disorders including hematologic conditions, immunodeficiencies, and genetic disorders.

  16. Proteomic analysis by iTRAQ in red claw crayfish, Cherax quadricarinatus, hematopoietic tissue cells post white spot syndrome virus infection.

    Science.gov (United States)

    Jeswin, Joseph; Xie, Xiao-Lu; Ji, Qiao-Lin; Wang, Ke-Jian; Liu, Hai-Peng

    2016-03-01

    To elucidate proteomic changes of Hpt cells from red claw crayfish, Cherax quadricarinatus, we have carried out isobaric tags for relative and absolute quantitation (iTRAQ) of cellular proteins at both early (1 hpi) and late stage (12 hpi) post white spot syndrome virus (WSSV) infection. Protein database search revealed 594 protein hits by Mascot, in which 17 and 30 proteins were present as differentially expressed proteins at early and late viral infection, respectively. Generally, these differentially expressed proteins include: 1) the metabolic process related proteins in glycolysis and glucogenesis, DNA replication, nucleotide/amino acid/fatty acid metabolism and protein biosynthesis; 2) the signal transduction related proteins like small GTPases, G-protein-alpha stimulatory subunit, proteins bearing PDZ- or 14-3-3-domains that help holding together and organize signaling complexes, casein kinase I and proteins of the MAP-kinase signal transduction pathway; 3) the immune defense related proteins such as α-2 macroglobulin, transglutaminase and trans-activation response RNA-binding protein 1. Taken together, these protein information shed new light on the host cellular response against WSSV infection in a crustacean cell culture. PMID:26845698

  17. Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis

    International Nuclear Information System (INIS)

    To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 oC (nonpermissive) to 32 oC (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of ∼40 kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis

  18. Proteomic Identification of Nrf2-Mediated Phase II Enzymes Critical for Protection of Tao Hong Si Wu Decoction against Oxygen Glucose Deprivation Injury in PC12 Cells

    Directory of Open Access Journals (Sweden)

    Hong-yi Qi

    2014-01-01

    Full Text Available Chinese herbal medicine formula Tao Hong Si Wu decoction (THSWD is traditionally used in China for cerebrovascular diseases. However, the molecular mechanisms of THSWD associated with the cerebral ischemia reperfusion injury are largely unknown. The current study applied the two-dimensional gel electrophoresis-based proteomics to investigate the different protein profiles in PC12 cells with and without the treatment of THSWD. Twenty-six proteins affected by THSWD were identified by MALDI-TOF mass spectrometry. Gene ontology analysis showed that those proteins participated in several important biological processes and exhibited diverse molecular functions. In particular, six of them were found to be phase II antioxidant enzymes, which were regulated by NF-E2-related factor-2 (Nrf2. Quantitative PCR further confirmed a dose-dependent induction of the six phase II enzymes by THSWD at the transcription level. Moreover, the individual ingredients of THSWD were discovered to synergistically contribute to the induction of phase II enzymes. Importantly, THSWD’s protection against oxygen-glucose deprivation-reperfusion (OGD-Rep induced cell death was significantly attenuated by antioxidant response element (ARE decoy oligonucleotides, suggesting the protection of THSWD may be likely regulated at least in part by Nrf2-mediated phase II enzymes. Thus, our data will help to elucidate the molecular mechanisms underlying the neuroprotective effect of THSWD.

  19. CELL-SELEX: Novel Perspectives of Aptamer-Based Therapeutics

    Directory of Open Access Journals (Sweden)

    Hans P. Wendel

    2008-04-01

    Full Text Available Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

  20. Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

    Directory of Open Access Journals (Sweden)

    Li Xing'an

    2010-08-01

    Full Text Available Abstract Background Cooperation of constituents of the ubiquitin proteasome system (UPS with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins incl