这项研究最初发表在分子细胞蛋白质组学。索尔多米和染色质功能域Bonaldi T的蛋白质组学研究揭示新型增效作用之间鲜明的异染色质成分的MCP。 2013; 12：64-80。 ©美国社会生物化学与分子生物学。我们感谢罗伯塔Noberini（技术和IEO的意大利学院，意大利）的手稿的批判性阅读。结核病工作是由来自乔瓦尼Armenise哈佛基金会职业发展计划，意大利癌症研究协会和健康的意大利外交部资助。 MS工作是由一个FIRC奖学金支持。

<ol>
	<li>Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+structure:+a+repeating+unit+of+histones+and+DNA.">Chromatin structure: a repeating unit of histones and DNA.</a> Science. 184, 868-871 (1974).</li><li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Bannister, A. J., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chromatin+by+histone+modifications.">Regulation of chromatin by histone modifications.</a> Cell Res. 21, 381-395 (2011).</li><li>Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D., Patel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+chromatin-binding+modules+interpret+histone+modifications:+lessons+from+professional+pocket+pickers.">How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers.</a> Nat Struct Mol Biol. 14, 1025-1040 (2007).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Spotswood, H. T., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increasingly+complex+code.">An increasingly complex code.</a> J Clin Invest. 110, 577-582 (2002).</li><li>Soldi, M., Cuomo, A., Bremang, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+proteomics+for+the+analysis+of+chromatin+structure+and+dynamics.">Mass spectrometry-based proteomics for the analysis of chromatin structure and dynamics.</a> Int J Mol Sci. 14, 5402-5431 (2013).</li><li>Sidoli, S., Cheng, L., 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=Proteomics+in+chromatin+biology+and+epigenetics:+Elucidation+of+post-translational+modifications+of+histone+proteins+by+mass+spectrometry.">Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry.</a> J Proteomics. 75, 3419-3433 (2012).</li><li>Britton, L. M., Gonzales-Cope, M., Zee, B. M., 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=Breaking+the+histone+code+with+quantitative+mass+spectrometry.">Breaking the histone code with quantitative mass spectrometry.</a> Expert Rev Proteomics. 8, 631-643 (2011).</li><li>Taverna, S. D., 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=Long-distance+combinatorial+linkage+between+methylation+and+acetylation+on+histone+H3+N+termini.">Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini.</a> Proc Natl Acad Sci U S A. 104, 2086-2091 (2007).</li><li>Garcia, B. A., Shabanowitz, J., Hunt, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+histones+and+their+post-translational+modifications+by+mass+spectrometry.">Characterization of histones and their post-translational modifications by mass spectrometry.</a> Curr Opin Chem Biol. 11, 66-73 (2007).</li><li>Villar-Garea, A., Imhof, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+histone+modifications.">The analysis of histone modifications.</a> Biochim Biophys Acta. 1764, 1932-1939 (2006).</li><li>Plazas-Mayorca, M. D., 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=One-pot+shotgun+quantitative+mass+spectrometry+characterization+of+histones.">One-pot shotgun quantitative mass spectrometry characterization of histones.</a> J Proteome Res. 8, 5367-5374 (2009).</li><li>Ohta, 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=The+protein+composition+of+mitotic+chromosomes+determined+using+multiclassifier+combinatorial+proteomics.">The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.</a> Cell. 142, 810-821 (2010).</li><li>Vermeulen, M., 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=Selective+anchoring+of+TFIID+to+nucleosomes+by+trimethylation+of+histone+H3+lysine+4.">Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4.</a> Cell. 131, 58-69 (2007).</li><li>Vermeulen, M., 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+interaction+proteomics+and+genome-wide+profiling+of+epigenetic+histone+marks+and+their+readers.">Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers.</a> Cell. 142, 967-980 (2010).</li><li>Bartke, 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=Nucleosome-interacting+proteins+regulated+by+DNA+and+histone+methylation.">Nucleosome-interacting proteins regulated by DNA and histone methylation.</a> Cell. 143, 470-484 (2010).</li><li>Dejardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins+associated+with+specific+genomic+Loci.">Purification of proteins associated with specific genomic Loci.</a> Cell. 136, 175-186 (2009).</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>Soldi, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proteomic+investigation+of+chromatin+functional+domains+reveals+novel+synergisms+among+distinct+heterochromatin+components.">The proteomic investigation of chromatin functional domains reveals novel synergisms among distinct heterochromatin components.</a> Mol Cell Proteomics. 12, 764-780 (2013).</li><li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes.">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nat Protoc. 1, 2856-2860 (2006).</li><li>Shevchenko, A., Wilm, M., Vorm, O., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+sequencing+of+proteins+silver-stained+polyacrylamide+gels.">Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.</a> Anal Chem. 68, 850-858 (1996).</li><li>Bonaldi, T., Regula, J. T., Imhof, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+mass+spectrometry+for+the+analysis+of+histone+modifications.">The use of mass spectrometry for the analysis of histone modifications.</a> Methods Enzymol. 377, 111-130 (2004).</li><li>Cuomo, A., Moretti, S., Minucci, S., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SILAC-based+proteomic+analysis+to+dissect+the+&amp;#34;histone+modification+signature&amp;#34;+of+human+breast+cancer+cells.">SILAC-based proteomic analysis to dissect the &amp;#34;histone modification signature&amp;#34; of human breast cancer cells.</a> Amino Acids. 41, 387-399 (2011).</li><li>Rappsilber, J., Mann, M., Ishihama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+micro-purification,+enrichment,+pre-fractionation+and+storage+of+peptides+for+proteomics+using+StageTips.">Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.</a> Nat Protoc. 2, 1896-1906 (2007).</li><li>Rappsilber, J., Ishihama, Y., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stop+and+go+extraction+tips+for+matrix-assisted+laser+desorption/ionization,+nanoelectrospray,+and+LC/MS+sample+pretreatment+in+proteomics.">Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.</a> Anal Chem. 75, 663-670 (2003).</li><li>Olsen, J. V., Ong, S. E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+cleaves+exclusively+C-terminal+to+arginine+and+lysine+residues.">Trypsin cleaves exclusively C-terminal to arginine and lysine residues.</a> Mol Cell Proteomics. 3, 608-614 (2004).</li><li>Jung, H. R., Pasini, D., Helin, K., 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=Quantitative+mass+spectrometry+of+histones+H3.2+and+H3.3+in+Suz12-deficient+mouse+embryonic+stem+cells+reveals+distinct,+dynamic+post-translational+modifications+at+Lys-27+and+Lys-36.">Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36.</a> Mol Cell Proteomics. 9, 838-850 (2010).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MaxQuant+enables+high+peptide+identification+rates,+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification.">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nat Biotechnol. 26, 1367-1372 (2008).</li><li>Cox, J., 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=Andromeda:+a+peptide+search+engine+integrated+into+the+MaxQuant+environment.">Andromeda: a peptide search engine integrated into the MaxQuant environment.</a> J Proteome Res. 10, 1794-1805 (2011).</li><li>Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., Jenuwein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+histone+H3+lysine+9+creates+a+binding+site+for+HP1+proteins.">Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.</a> Nature. 410, 116-120 (2001).</li><li>Bannister, A. J., 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=Selective+recognition+of+methylated+lysine+9+on+histone+H3+by+the+HP1+chromo+domain.">Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.</a> Nature. 410, 120-124 (2001).</li><li>Ram, 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=Combinatorial+patterning+of+chromatin+regulators+uncovered+by+genome-wide+location+analysis+in+human+cells.">Combinatorial patterning of chromatin regulators uncovered by genome-wide location analysis in human cells.</a> Cell. 147, 1628-1639 (2011).</li><li>Barski, 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129, 823-837 (2007).</li><li>Beck, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+in+epigenetic+research.">Mass spectrometry in epigenetic research.</a> Methods Mol Biol. 593, 263-282 (2010).</li><li>Ernst, J., Kellis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+characterization+of+chromatin+states+for+systematic+annotation+of+the+human+genome.">Discovery and characterization of chromatin states for systematic annotation of the human genome.</a> Nat Biotechnol. 28, 817-825 (2010).</li><li>Tipton, J. D., 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=Analysis+of+intact+protein+isoforms+by+mass+spectrometry.">Analysis of intact protein isoforms by mass spectrometry.</a> J Biol Chem. 286, 25451-25458 (2011).</li><li>Young, N. 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=High+throughput+characterization+of+combinatorial+histone+codes.">High throughput characterization of combinatorial histone codes.</a> Mol Cell Proteomics. 8, 2266-2284 (2009).</li><li>Boyne, M. T., Pesavento, J. J., Mizzen, C. A., Kelleher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+characterization+of+human+histones+in+the+H2A+gene+family+by+top+down+mass+spectrometry.">Precise characterization of human histones in the H2A gene family by top down mass spectrometry.</a> J Proteome Res. 5, 248-253 (2006).</li></ol>

没有利益冲突的声明。

我们最近已描述ChroP，定量策略染色质中的蛋白质组分的大规模表征。 ChroP结合的表观遗传领域使用两种互补的方法，芯片和质谱，从自己的优势中获利，克服各自的局限性。沉淀耦合到深度测序技术（ChIP-SEQ）允许的组蛋白修饰在核小体少35的分辨率的全基因组的映射。虽然便于其敏感性，基于抗体的测定是在自己有能力区分类似的修改和解剖的组蛋白密码36组合方面的限制。另?...

染色质是涉及为所有的DNA介导的过程主模板一个高度动态的核蛋白复合物。核小体是染色质的基本重复单元和由含各规范组蛋白H2A，H2B，H3和H4，在其周围147 bp的DNA被包裹1,2两分子的蛋白质八聚体核心的。所有核心组蛋白的结构为球状结构域和一个灵活的N端“尾巴”突出了核小体之外。一种用于调节染色质结构和动力学的主要机制是基于共价键的翻译后修饰（翻译后修饰），其主要发生在N-末端组蛋白3,4。组蛋白修饰可以通过改变高阶染色质结构，通过改变之间的组蛋白-DNA或核小体之间的接触，并从而控制DNA结合蛋白（ 顺机制）的可访问功能，或通过作为dockin克位点的调节蛋白，无论是作为单个单元，或者嵌入在多聚体复合物。这样调节蛋白可以发挥它们的功能以不同的方式：通过直接调节基因表达（ 即 TAF蛋白），或通过改变核小体定位（ 即染色质重塑复合物），或者通过修改其他组蛋白残基（ 即蛋白质具有甲基转移或乙酰基转移酶活性）（ 反式机制）5。观察到在某些具体的染色质基因座不同的PTM模式群集导致了阐述这一假设在不同地点的不同修饰可协同作用以生成分子编码介导相关的DNA的功能状态。在“组蛋白密码假说”已经获得了很大的共识在多年，但它的实验验证已受困于技术限制6,7。 质谱（MS）为基础的蛋白质组学已成为一个功能强大的工具来映射组蛋白修饰模式和表征染色质结合蛋白8。 MS检测的变形作为肽的实验和理论质量之间的特定Δmass。在个人组蛋白的水平，微软提供了一个公正和全面的方法来映射翻译后修饰，允许其中9-14新的修改和揭示interplays的检测。 在最近几年，已经开发了多种策略来剖析染色质的蛋白质组合物，包括完整的有丝分裂染色体15，可溶性HPTM结合蛋白16-18的标识和特定染色质区域的分离和分析的表征（ 即端粒）19,20。然而，组蛋白翻译后修饰，变体和染色质相关蛋白之间的位点特异性的协同作用的研究仍然是不完整。在这里，我们描述了一种新的方法，命名为ChroP（染色体晨报P roteomics）21，我们已经开发了能够有效表征功能不同的染色质域。这种方法适应染色质免疫沉淀技术（ChIP），在表观遗传学研究中使用一套行之有效的方案，为充实样品的有效质谱为基础的蛋白质组学分析。我们已经开发出两种不同的协议，这取决于所用的输入和由MS处理的问题染色质的类型;具体为：1）芯片消化MNase不固定本地染色质是用来净化单核和解剖合作关联hPTMs（N-ChroP）; 2）交联染色质碎片通过超声处理芯片采用的是与SILAC为主interactomics策略来描述所有共同丰富的染色质结合蛋白（X-ChroP）的组合。我们举例说明这里的N-和X-ChroP的丰富和研究异，采用H3K9me3为诱饵进行免疫沉淀步骤的结合。使用ChroP的可扩展研究无论是不同的区域染色质上，或变化过渡到一个不同的功能状态，在同一区域内的染色质组成，从而铺平了道路表观遗传学中的各种应用。

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">DMEM&#160;</td>
			<td style="width:145px;">Lonza</td>
			<td style="width:119px;">BE12-614F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">FBS</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">SILAC DMEM</td>
			<td style="width:145px;">M-Medical</td>
			<td style="width:119px;">FA30E15086</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Dialyzed FBS</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:119px;">26400-044</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Lysine 0 (12C6 14N2 L-lysine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">L8662</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Arginine 0 (12C6 14N4 L-arginine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">A6969</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lysine 8 (13C6 15N2 L-lysine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td>68041</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Arginine 10 (13C6 15N4 L-arginine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td>608033</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Micrococcal Nuclease</td>
			<td style="width:145px;">Roche</td>
			<td style="width:119px;">10 107 921 001</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:271px;">Complete, EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td style="width:145px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Spectra/Por 3 dialysis tubing, 3.5K MWCO, 18mm flat width, 50 foot length</td>
			<td style="width:145px;">Spectrumlabs</td>
			<td style="width:119px;">132720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">QIAquick PCR purification kit</td>
			<td style="width:145px;">QIAGEN</td>
			<td style="width:119px;">28104</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Anti-Histone H3 tri-methylated K9-ChIP grade</td>
			<td>Abcam</td>
			<td>ab8898</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Histone H3 peptide tri-methyl K9&#160;</td>
			<td>Abcam</td>
			<td style="width:119px;">ab1773</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td style="width:119px;">100.04D</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">NuPAGE Novex 4-12%&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Bis-Tris Gel&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>NP0335BOX</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Colloidal Blue Staining Kit</td>
			<td>Invitrogen&#160;</td>
			<td>LC6025</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LDS Sample Buffer</td>
			<td>Invitrogen&#160;</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Formaldheyde</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Aceti anhydride-d6</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">175641-1G</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Formic Acid</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">94318-50ML-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Iodoacetamide &#8805;99% (HPLC), crystalline</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">I6125</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">DL-Dithiothreitol</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">43815</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Sequencing Grade Modified Trypsin, Frozen 100ug (5 &#215; 20&#956;g)</td>
			<td>Promega</td>
			<td style="width:119px;">V5113</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Nanospray OD 360&#956;m x ID 75&#956;m, tips ID 8&#956;m uncoated Pk 5</td>
			<td style="width:145px;">Microcolumn Srl</td>
			<td style="width:119px;">FS360-75-8-N-5-C15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">&#160; ReproSil-Pur 120 C18-AQ, 3 &#181;m&#160;&#160; 15 % C</td>
			<td style="width:145px;">Dr. Maisch GmbH</td>
			<td style="width:119px;">r13.aq.</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">C18 extraction disk, 47 mm</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">1&#8203;2&#8203;1&#8203;4&#8203;5&#8203;0&#8203;0&#8203;4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Carbon extraction disk, 47 mm</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">12145040</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Cation extraction disk</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">66889-U</td>
			<td></td>
		</tr>
	</tbody>
</table>

the chrop approach combines chip and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin

染色质是一种高动态核蛋白复合物由DNA和蛋白质，其控制各个DNA依赖性过程。在特定区域的染色质结构和功能是由组蛋白的翻译后修饰（hPTMs）和变体，染色质结合蛋白，包括转录因子，和DNA甲基化的局部富集调节。染色组合物中的不同的功能区的蛋白质组分析的方法已经被缺乏有效的协议，以丰富这些域在适当的纯度和量为后续的深入分析物的质谱（MS）到目前为止妨碍。我们在这里介绍了新设计的染色质蛋白组学策略，命名ChroP（CHRO晨报P roteomics），由此制备型染色质免疫沉淀是用来隔离不同的染色质区域，其特征在hPTMs方面，变体和共同相关联的非histonic蛋白质，是通过MS进行分析。我们说明他重新ChroP的转录沉默的异染色质区域的富集和分析，其标志是三甲基化的组蛋白H3赖氨酸9的存在成立。所取得的结果证明ChroP在彻底表征异蛋白质组，并证明它作为理解如何染色质的独特的蛋白质决定簇相互作用和协同效应，建立基因座的特定结构和功能结构的有力的分析策略的潜力。

染色质免疫沉淀法是用于分析沿基因组中的蛋白或组蛋白修饰的定位一个强大的技术。在蛋白质组学当量，芯片随后基于MS的蛋白质组学识别定性和定量的hPTMs，组蛋白变体与染色质结合蛋白是与感兴趣的修饰或蛋白免疫沉淀一起，作为“诱饵”。在N-ChroP方法中，在图1A中，原生芯片，其中染色质消化MNase（ 图1B）所述，作为输入到从散装染色质独特的功能域净化。消化染...

Watch this Scientific Journal Video about The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin at JoVE.com

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

通过结合本地和交联染色质免疫沉淀高分辨率质谱，ChroP方法使解剖的组蛋白修饰，变体和非histonic协同作用的蛋白质在功能上不同的染色质域的复合蛋白质结构。

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

the-chrop-approach-combines-chip-mass-spectrometry-to-dissect-locus

Research

JoVE Journal

Biology

18.1K 次观看.  European Institute of Oncology. 通过结合本地和交联染色质免疫沉淀高分辨率质谱，ChroP方法使解剖的组蛋白修饰，变体和非histonic协同作用的蛋白质在功能上不同的染色质域的复合蛋白质结构。 

视频: The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...