Name: quantitative analysis of chromatin proteomes in disease
Uploaded: 2012-12-28T12:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Chromatin Proteomes in Disease at JoVE.com

Die Vondriska Labor ist durch Zuschüsse aus dem National Heart, Lung and Blood Institute der NIH und der Laubisch Endowment an der UCLA unterstützt. EM ist Empfänger der Jennifer S. Buchwald Graduate Fellowship für Physiologie an der UCLA; HC ist der Empfänger einer American Heart Association Pre-Doc-Stipendium; MP ist der Empfänger einer NIH Ruth Kirschstein Post-Doktoranden-Stipendium und SF ist der Empfänger von ein NIH K99 Award.

Die experimentelle Workflow enthält sieben großen Schritten (Abbildung 1). Bei allen Arbeiten, Proben, die auf dem Massenspektrometer laufen soll, sollte der Experimentator tragen Laborkittel, Handschuhe und Haarnetz und kümmern uns um Verunreinigung durch Staub und persönliche Abbau von Keratin zu vermeiden. Ein. Herz Homogenisierung und Reaktorsicherheit Isolation Mäuseherzen werden homogenisiert und eine intakte Nuklei isoliert Pellet wird ...

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	<li>Li, F., Wang, X., Capasso, J. M., Gerdes, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+transition+of+cardiac+myocytes+from+hyperplasia+to+hypertrophy+during+postnatal+development.">Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development.</a> J Mol Cell Cardiol. 28, 1737-1746 (1996).</li><li>Rumyantsev, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrelations+of+the+proliferation+and+differentiation+processes+during+cardiact+myogenesis+and+regeneration.">Interrelations of the proliferation and differentiation processes during cardiact myogenesis and regeneration.</a> Int Rev. Cytol. 51, 186-273 (1977).</li><li>Olson, E. N., Schneider, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sizing+up+the+heart:+development+redux+in+disease.">Sizing up the heart: development redux in disease.</a> Genes Dev. 17, 1937-1956 (2003).</li><li>Molkentin, J. D., Dorn, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+signaling+pathways+that+regulate+cardiac+hypertrophy.">Cytoplasmic signaling pathways that regulate cardiac hypertrophy.</a> Annu Rev. Physiol. 63, 391-426 (2001).</li><li>Fishman, M. C., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fashioning+the+vertebrate+heart:+earliest+embryonic+decisions.">Fashioning the vertebrate heart: earliest embryonic decisions.</a> Development. 124, 2099-2117 (1997).</li><li>Rajabi, M., Kassiotis, C., Razeghi, P., Taegtmeyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Return+to+the+fetal+gene+program+protects+the+stressed+heart:+a+strong+hypothesis.">Return to the fetal gene program protects the stressed heart: a strong hypothesis.</a> Heart Fail Rev. 12, 331-343 (2007).</li><li>Roger, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2011+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2011 update: a report from the American Heart Association.</a> Circulation. 123, e18-e209 (2011).</li><li>Schirmer, E. C., Florens, L., Guan, T., Yates, J. R., Gerace, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+membrane+proteins+with+potential+disease+links+found+by+subtractive+proteomics.">Nuclear membrane proteins with potential disease links found by subtractive proteomics.</a> Science. 301, 1380-1382 (2003).</li><li>Lambert, J. P., Mitchell, L., Rudner, A., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+proteomics+approach+for+the+discovery+of+chromatin-associated+protein+networks.">A novel proteomics approach for the discovery of chromatin-associated protein networks.</a> Mol. Cell Proteomics. 8, 870-882 (2009).</li><li>Malik, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+and+lamin-dependent+targeting+of+novel+transmembrane+proteins+in+the+nuclear+envelope.">Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope.</a> Cell Mol Life Sci. 67, 1353-1369 (2010).</li><li>Tweedie-Cullen, R. Y., Reck, J. M., Mansuy, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+post-translational+modifications+on+synaptic,+nuclear,+and+histone+proteins+in+the+adult+mouse+brain.">Comprehensive mapping of post-translational modifications on synaptic, nuclear, and histone proteins in the adult mouse brain.</a> J. Proteome Res. 8, 4966-4982 (2009).</li><li>Chu, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+chromatin+proteomics+identifies+evolutionarily+conserved+fertility+factors.">Sperm chromatin proteomics identifies evolutionarily conserved fertility factors.</a> Nature. 443, 101-105 (2006).</li><li>Uchiyama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+human+metaphase+chromosomes.">Proteome analysis of human metaphase chromosomes.</a> J. Biol. Chem. 280, 16994-17004 (2005).</li><li>Franklin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+compartments+of+cardiac+nuclei+exhibit+distinct+proteomic+anatomy.">Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy.</a> Mol. Cell Proteomics. 10, (2011).</li><li>Kislinger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+survey+of+organ+and+organelle+protein+expression+in+mouse:+combined+proteomic+and+transcriptomic+profiling.">Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling.</a> Cell. 125, 173-186 (2006).</li><li>Moore, D. H., Ruska, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+study+of+mammalian+cardiac+muscle+cells.">Electron microscope study of mammalian cardiac muscle cells.</a> J. Biophys Biochem. Cytol. 3, 261-268 (1957).</li><li>Anversa, P., Vitali-Mazza, L., Visioli, O., Marchetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+cardiac+hypertrophy:+a+quantitative+ultrastructural+study+in+the+compensatory+stage.">Experimental cardiac hypertrophy: a quantitative ultrastructural study in the compensatory stage.</a> J. Mol. Cell Cardiol. 3, 213-227 (1971).</li><li>Franklin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+compartments+of+cardiac+nuclei+exhibit+distinct+proteomic+anatomy.">Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy.</a> Mol Cell. Proteomics. 10, (2011).</li><li>Paulsson, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+regulation+of+calsarcin-1+during+pressure+overload-induced+cardiac+hypertrophy.">Post-translational regulation of calsarcin-1 during pressure overload-induced cardiac hypertrophy.</a> Journal of molecular and cellular cardiology. 48, 1206-1214 (2010).</li><li>Franklin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+chromatin+proteome+in+disease+reveals+remodeling+principles+and+identifies+high+mobility+group+protein+b2+as+a+regulator+of+hypertrophic+growth.">Quantitative analysis of the chromatin proteome in disease reveals remodeling principles and identifies high mobility group protein b2 as a regulator of hypertrophic growth.</a> Mol Cell Proteomics. 11, (2012).</li><li>Gramolini, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+profiling+of+a+phospholamban+mutant+mouse+model+of+dilated+cardiomyopathy+reveals+progressive+intracellular+stress+responses.">Comparative proteomics profiling of a phospholamban mutant mouse model of dilated cardiomyopathy reveals progressive intracellular stress responses.</a> Mol Cell Proteomics. 7, 519-533 (2008).</li><li>Singh, H., Warburton, S., Vondriska, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+to+Identify+Proteins+Interacting+with+P2X2+Ligand-Gated+Cation+Channels.">Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels.</a> J. Vis. Exp. (27), e1178 (2009).</li><li>Park, S. K., Venable, J. D., Xu, T., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+software+tool+for+mass+spectrometry-based+proteomics.">A quantitative analysis software tool for mass spectrometry-based proteomics.</a> Nat. Methods. 5, 319-322 (2008).</li><li>Lomenick, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+identification+using+drug+affinity+responsive+target+stability+(DARTS).">Target identification using drug affinity responsive target stability (DARTS).</a> Proc. Natl. Acad. Sci. U. S. A. 106, 21984-21989 (2009).</li><li>Kamleh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+profiling+using+Orbitrap+Fourier+transform+mass+spectrometry+with+hydrophilic+interaction+chromatography:+a+method+with+wide+applicability+to+analysis+of+biomolecules.">Metabolomic profiling using Orbitrap Fourier transform mass spectrometry with hydrophilic interaction chromatography: a method with wide applicability to analysis of biomolecules.</a> Rapid Commun. Mass Spectrom. 22, 1912-1918 (2008).</li><li>Searle, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold:+a+bioinformatic+tool+for+validating+MS/MS-based+proteomic+studies.">Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies.</a> Proteomics. 10, 1265-1269 (2010).</li><li>Murray, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Basic+Proteins+of+Cell+Nuclei.">The Basic Proteins of Cell Nuclei.</a> Annu Rev. Biochem. 34, 209-246 (1965).</li><li>Johns, E. W., Phillips, D. M., Simson, P., Butler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+fractionations+of+arginine-rich+histones+from+calf+thymus.">Improved fractionations of arginine-rich histones from calf thymus.</a> Biochem. J. 77, 631-636 (1960).</li><li>Rodriguez-Collazo, P., Leuba, S. H., Zlatanova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+methods+for+purification+of+histones+from+cultured+mammalian+cells+with+the+preservation+of+their+native+modifications.">Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.</a> Nucleic Acids Res. 37, e81 (2009).</li><li>Kuehl, L., Salmond, B., Tran, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentrations+of+high-mobility-group+proteins+in+the+nucleus+and+cytoplasm+of+several+rat+tissues.">Concentrations of high-mobility-group proteins in the nucleus and cytoplasm of several rat tissues.</a> J. Cell Biol. 99, 648-654 (1984).</li><li>Gronow, M., Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+and+separation+of+the+non-histone+proteins+of+rat+liver+nuclei.">Rapid isolation and separation of the non-histone proteins of rat liver nuclei.</a> FEBS Lett. 15, 340-344 (1971).</li><li>Mischke, B. G., Ward, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+tissue+specificity+of+chromatin-associated+proteins+in+Vicia+faba.">Isolation and tissue specificity of chromatin-associated proteins in Vicia faba.</a> Can J. Biochem. 53, 91-95 (1975).</li><li>Andersen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+proteomic+analysis+of+the+human+nucleolus.">Directed proteomic analysis of the human nucleolus.</a> Curr. Biol. 12, 1-11 (2002).</li><li>Zhao, Y., Jensen, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification-specific+proteomics:+strategies+for+characterization+of+post-translational+modifications+using+enrichment+techniques.">Modification-specific proteomics: strategies for characterization of post-translational modifications using enrichment techniques.</a> Proteomics. 9, 4632-4641 (2009).</li><li>Ahrne, E., Muller, M., Lisacek, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unrestricted+identification+of+modified+proteins+using+MS/MS.">Unrestricted identification of modified proteins using MS/MS.</a> Proteomics. 10, 671-686 (2010).</li><li>Young, N. L., Plazas-Mayorca, M. D., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems-wide+proteomic+characterization+of+combinatorial+post-translational+modification+patterns.">Systems-wide proteomic characterization of combinatorial post-translational modification patterns.</a> Expert Rev. Proteomics. 7, 79-92 (2010).</li><li>Guthals, A., Bandeira, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+identification+by+tandem+mass+spectrometry+with+alternate+fragmentation+modes.">Peptide identification by tandem mass spectrometry with alternate fragmentation modes.</a> Mol. Cell Proteomics. , (2012).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protease+for+'middle-down'+proteomics.">A protease for 'middle-down' proteomics.</a> Nat. Methods. , (2012).</li><li>Tran, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+intact+protein+isoforms+in+discovery+mode+using+top-down+proteomics.">Mapping intact protein isoforms in discovery mode using top-down proteomics.</a> Nature. 480, 254-258 (2011).</li></ol>

Zwei Hauptverfahren für nukleare Isolierung wurden zuvor bewertet: 27 eine ist die Technik der Behrens Homogenisieren lyophilisierten Gewebe in einem nicht-wässrigen Lösungsmittel und das zweite, eine Modifikation der wir hier von Homogenisier Gewebe in einer wäßrigen Saccharose / Salzlösung gefolgt durch differentielle oder Dichtegradientenzentrifugation. Subfraktionierung der Kerne durch saure Extraktion von Gewebeproben ist ein wichtiges Werkzeug für die Untersuchung Chr...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Dulbeco Modified Eagle Medium </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> Protease Pellet </td><td> Roche </td><td> 04 693 159 001 </td></tr><tr><td> 100 um Sieb </td><td> BD Falcon </td><td> 352360 </td></tr><tr><td> Ultracut Ultramikrotom </td><td> Reichert </td><td></td></tr><tr><td> 100CX Transmission Electron Mikroskop </td><td> JEOL USA, Inc. </td><td></td></tr><tr><td> Pirol </td><td> BioRad </td><td> 161-0496 </td></tr><tr><td> Histon H2A-Antikörper </td><td> Santa Cruz </td><td> sc-8648 </td></tr><tr><td> Nucleoporin p62 Antikörper </td><td> BD Biosciences </td><td> 610498 </td></tr><tr><td> Adenin nucleoFlut transporter Antikörper </td><td> Santa Cruz </td><td> sc-9299 </td></tr><tr><td> BiP-Antikörper </td><td> Santa Cruz </td><td> sc-1050 </td></tr><tr><td> Tubulin-Antikörper </td><td> Sigma </td><td> T1568 </td></tr><tr><td> Histone H3 Antikörper </td><td> Abcam </td><td> ab1791 </td></tr><tr><td> Fibrillarin Antikörper </td><td> Cell Signaling </td><td> C12C3 </td></tr><tr><td> SNRP70 Antikörper </td><td> Abcam </td><td> ab51266 </td></tr><tr><td> E2F-1-Antikörpers </td><td> Thermo Fisher </td><td> MS-879 </td></tr><tr><td> Retinoblastoma Antikörper </td><td> BD Biosciences </td><td> 554136 </td></tr><tr><td> Hypoxie-induzierbaren Faktor 1-Antikörpers </td><td> Novus Biologicals </td><td> NB100-469 </td></tr><tr><td> BCA-Protein-Assay </td><td> Thermo Scientific </td><td> 23227 </td></tr><tr><td> Umkehrphasensäule </td><td> New Objektive </td><td> PFC7515-B14-10 </td></tr><tr><td> BioWorks Browser </td><td> Thermo Scientific </td><td></td></tr></tbody></table>

quantitative analysis of chromatin proteomes in disease

Im Zellkern befinden die Proteome, deren Funktionen auf das engste mit der Genregulation verknüpft. Adulten Kardiomyozyten Kerne sind einzigartig wegen des hohen Anteils an zweikerniger Zellen, 1 die überwiegend heterochromatischen Zustand der DNA, und das nicht-teilenden Art der Herzmuskelzellen, die Erwachsenen Kerne macht in einem permanenten Zustand der Interphase. 2 Transkriptionelle Regulation während der Entwicklung und Krankheiten wurden auch in diesem Organ, 3-5 studiert, aber was bleibt relativ unerforscht ist die Rolle der nuklearen Proteine, die für DNA-Verpackung und Ausdruck gespielt, und wie diese Proteine ​​Veränderungen in der transkriptionellen Programme, die während Krankheit auftreten steuern. 6 In den Industrieländern Welt, Herzkrankheiten die häufigste Ursache der Sterblichkeit für Männer und Frauen. 7 Insight, wie nukleare Proteine ​​zusammen, um das Fortschreiten dieser Krankheit zu regeln ist entscheidend für die Förderung der aktuellen Behandlung optionen. Die Massenspektrometrie ist das ideale Werkzeug für die Bewältigung dieser Fragen, wie es für eine unvoreingenommene Annotation des nuklearen Proteom und relative Quantifizierung ermöglicht, wie die Fülle dieser Proteine ​​verändert sich mit Krankheit. Zwar gibt es mehrere proteomischen Studien für Säugetier nuklearen Proteinkomplexe, 8-13 bis vor kurzem gab es 14 nur eine Studie, die die kardiale nuklearen Proteom, und es als das gesamte Kern, anstatt Erkundung des Proteoms auf der Ebene der Fächer Nuklearteilbild . 15 In einem großen Teil ist dies Mangel an Arbeit wegen der Schwierigkeit der Isolierung Herz Kerne. Cardiac Kerne treten innerhalb einer starren und dichten Aktin-Myosin Vorrichtung, an die sie über mehrere Erweiterungen aus dem endoplasmatischen Retikulum verbunden sind, in dem Umfang, Myozyten Kontraktion verändert ihre Gesamtform. 16 Zusätzlich Kardiomyozyten 40% Mitochondrien Vol. 17, die necessitätes Anreicherung des Kernes von den anderen Organellen. Hier beschreiben wir ein Protokoll für kardiale Urananreicherung und weitere Fraktionierung in biologisch relevanten Fächern. Darüber hinaus haben wir detailliert Verfahren zur markierungsfreien quantitativen massenspektrometrischen Dissektion dieser Fraktionen-Techniken zugänglich in vivo Experimente in verschiedenen Tiermodellen und Organsysteme, wo metabolische Markierung nicht durchführbar ist.

Abbildung 4 verdeutlicht den Nutzen dieser Form der relativen Quantifizierung. Gezeigt im linken Fenster sind die einzelnen monoisotopic Peptidpeaks (überlagert von verschiedenen Mäusen), die als Angehörige der Protein HMGB1 (identifiziert über Datenbanksuche) bezeichnet wurden. Jeder Peak, im Wesentlichen eine extrahierte Ionenchromatographen für die gegebene Peptid, kommt aus einer anderen Maus. Die Gruppen repräsentieren drei verschiedene physiologische Zustände: basal, Herzhypertrophie und He...

Watch this Scientific Journal Video about Quantitative Analysis of Chromatin Proteomes in Disease at JoVE.com

Quantitative Analysis of Chromatin Proteomes in Disease

Advances in Massenspektrometrie haben die Hochdurchsatz-Analyse der Proteinexpression und Modifikation in einer Vielzahl von Geweben erlaubt. Kombiniert mit subzelluläre Fraktionierung und Krankheitsmodelle, quantitative Massenspektrometrie und der Bioinformatik neue Eigenschaften in biologischen Systemen offenbaren kann. Das hierin beschriebene Verfahren analysiert Chromatin-assoziierten Proteinen in der Einstellung von Herzerkrankungen und ist ohne weiteres auch auf andere<em&gt; In vivo</em&gt; Modelle menschlicher Erkrankungen.

Quantitative Analyse von Chromatin Proteome in Disease

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Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

Anwendung von Granger Kausalität Analyse des gerichteten funktionalen Zusammenhangs in der Alzheimer-Krankheit und Mild Cognitive Impairment

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in MeniÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¨re's Disease Ears and Normal Hearing Ears

In Vivo Morphometrische Analyse der menschlichen Hirnnerven mittels Kernspintomographie in der Menière Krankheit Ohren und Normal hörende Ohren

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRIÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚ÂApplication in Premanifest Huntington's Disease

Gesamte Gehirn Segmentierung und Change-Point-Analyse der anatomischen Gehirn MRI-Anwendung in Prämanifesten Huntington-Krankheit

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-aminopropionitrile-induced Aortic Aneurysm and Dissection

Quantitative Micro-CT-Analyse der Aortopathy in einem Mausmodell der β-Aminopropionitrile-induzierte Aortenaneurysma und Dissektion

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...