的Vondriska的补助金从国家心脏，肺和血液研究所，美国国立卫生研究院和Laubisch基金会在加州大学洛杉矶分校实验室的支持。 EM是收件人的詹妮弗学的布赫瓦尔德研究生奖学金，加州大学洛杉矶分校生理学，HC是收件人的美国心脏协会前博士生奖学金; MP是收件人的美国国立卫生研究院露丝基尔希斯坦博士后研究;和SF是收件人的NIH K99奖。

实验工作流包含七个主要步骤（ 图1）。对于任何有关的工作，将运行在质谱仪的样品，实验人员应穿白大褂，手套和头发网和照顾，以避免污染，灰尘和脱落的角质个人。 1。心同质化和核孤立小鼠心脏均化和一个完整的核颗粒中分离（ 图2）。 <ol><li>牺牲成年小鼠，切除心脏，在冰冷的PBS冲洗，并在冰上摇匀玻璃DOUNCE（我们优选从Fisher的威敦组织磨床，＃08-414-13A，但其它方法也同样出色的工作）含有2毫升裂解缓冲液（低渗溶液，其中差分溶解细胞膜的细胞器，包括细胞核）（10mM的Tris pH为7.5，15 mM氯化钠，0.15％体积/体积NONIDET P-40 [NP-40]在去离子水中加蛋白酶和磷酸我nhibitor混合物:10mM的丁酸钠，0.1 1mM苯甲磺酰氟[PMSF]，0.2毫摩尔的Na 3 VO 4，0.1mM的NaF和1罗氏蛋白酶pellet/10毫升-裂解缓冲液可以存储最多一周的在-20℃下）。 （ 注:我们不觉得有必要与PBS灌注的心，我们的质谱数据，GO分析鉴定的蛋白质为主心肌细胞的P-值3.8E-22]，而不是起源于血液污染[p值5.0 E-2]）。 </li><li>倒裂解液通过100微米的过滤器，并通过收集流量1.5 mL离心管中。在这一点上，除非特别指明，样品应保存在冰中。 </li><li> 4,000 rpm离心5分钟，在4℃下离心</li><li>除去上清液。 （这是胞质溶胶中，并应贮存于-80℃。包含溶解的线粒体。）沉淀重悬在200μl裂解缓冲液由捣碎。 （这是粗核丸）。 </li><li>用1毫升蔗糖的buff填充1.5毫升离心管中蔗糖24％重量/体积，pH为7.5的10mM Tris，和15mM NaCl在与蛋白酶的去离子水/磷酸酶抑制剂混合物（呃 - 蔗糖缓冲液应在使用当天新鲜）。轻轻层，将重新悬浮的颗粒蔗糖垫的顶部和离心机在5,000 rpm，10分钟，在4℃下</li><li>拆下顶部的薄膜上，以及蔗糖垫（包含膜）。冲洗剩余的沉淀用200μl冰冷的PBS / EDTA（1×PBS，用1mM EDTA）。 （这是细胞核沉淀和蛋白质印迹或电子显微镜中使用的，可以溶解或固定[见步骤4.1和4.2]量化富集。） </li></ol> 2。核质和洗涤剂提取的染色质分馏将粗核沉淀被分离成核质和洗涤剂提取染色质馏分，含有蛋白质与DNA松散联系。 <ol><li>捣碎颗粒从步骤1.6的洗涤剂在200μl提取缓冲液（20 mM HEPES的pH为7.6，7.5毫摩尔MgCl 2。 1 M尿素，30 mM氯化钠，0.2mM的EDTA，1％NP-40中的去离子水与蛋白酶/磷酸酶抑制剂混合物 - 洗涤剂提取缓冲液可以存储最多一周的在-20℃）。 </li><li>涡样品2次，每次10秒。放置在冰上10分钟。 </li><li>离心机以13,000 rpm离心5分钟，在4℃下</li><li>除去上清液。 （这是核质，并应贮存在-80℃下）。冲洗沉淀，用冰冷的PBS / EDTA。 （这是染色质的颗粒。） </li><li>捣碎沉淀中加入300μl的Tris，SDS，EDTA缓冲液（50mM的Tris pH值7.4，10mM的EDTA，1％SDS在去离子水中用蛋白酶/磷酸酶抑制剂混合物 - 的Tris，SDS，EDTA缓冲液可以保持长达1周-20°C）。 </li><li>超声波清洗3-6次，持续10秒，打破了DNA。保持样品在冰之间的超声处理。 </li><li>离心机以13,000 rpm离心5分钟，在4℃下保留上清液。 （上清液的洗涤剂提取染色质蛋白fraction和应保持在-80℃）。余下的颗粒应该是小的，并且可以被丢弃。 </li><li>如果使用的，而不是整个心脏的离体心肌细胞:分离新生大鼠心室肌细胞从出生后幼鼠1天，用酶消化，其次是文化的Dulbecco改良Eagle培养基（DMEM），10％胎牛血清（FBS），1％的胰岛素转亚硒酸钠（ITS），和1％青霉素。 24小时后，转移到无血清培养基（同上，但缺乏FBS）。收获细胞裂解缓冲液（1.1），并开始核分馏步骤1.3。 </li></ol> 3。酸萃取分馏 - DNA结合蛋白的富集一个单独的分馏丰富的蛋白质紧密地结合到DNA上，包括组蛋白。 <ol><li>重复步骤1.1-2.4。 </li><li>捣碎的染色质的颗粒在400微升0.4 N硫酸。涡删除团块。 </li><li>在4℃下孵育30分钟或overnIGHT而旋转。 </li><li>在16000 XG离心10分钟，在4°C，以消除核碎片。 </li><li>上清转移到一个新的管。 </li><li>三氯乙酸逐滴上清液中加入132μl。反转几次。在冰上孵育30分钟。 </li><li>在4℃下以16,000 xg离心10分钟，离心</li><li>弃去上清液。轻轻漂洗沉淀用冰冷的丙酮。 （这是组蛋白的颗粒。） </li><li>在4℃下以16,000 xg离心10分钟，离心</li><li>重复洗，步骤3.8-3.9。 </li><li>空气干燥沉淀。 </li><li>将沉淀重悬于100微升的Tris，SDS，EDTA缓冲液。通过加入1M Tris，设置pH至8。 （使用的1 M Tris股票，PH值不调整）。 </li><li>超声水浴中15分钟。冰水浴防止过热。 （这是萃取的酸部分，并应贮存于-80℃。） </li></ol> 4。纯度检查 Western印迹和电子显微镜确认成功富集的细胞核和其他细胞器的枯竭。要看到所有的下列质量控制分析的典型结果，请参见我们之前发表的。18 <ol><li>进行Western blot分析。探头的膜含有各馏分的组蛋白H2A nucleoporin的p62的（为核标记验证富集），和腺嘌呤核苷酸转运体，BiP和微管蛋白（作为线粒体标记，内质网的标记，和细胞骨架的标记分别验证纯度）。为了验证成功subfractionation的原子核，探头组蛋白H3或核仁纤维（洗涤剂和酸提取细胞核染色质和完整的）和SNRP70或E2F（核质）。 2002视网膜母细胞瘤或缺氧诱导因子-1（在洗涤剂中提取染色质丰富，酸提取部分）。额外的控制样品的HeLa细胞裂解液和整个心脏裂解液也可以运行在同一凝胶:准备HeLa细胞裂解液加入三，SDS，ED的控制TA缓冲液的培养皿中，用细胞刮刀收集样品。超声波清洗离心样品的步骤2.6-2.7。通过均质化的心脏在2ml缓冲液（20mM的Tris pH值7.4，150mM的NaCl，1mM的EDTA，1mM的EGTA，1％的Triton X-100，2.5 mM的焦磷酸钠，在去离子水中的1mM的磷酸甘油准备整个心脏溶解物控制用蛋白酶和磷酸酶抑制剂混合物）。超声波清洗和离心的步骤2.6-2.7。请参阅第5步中，用于制备蛋白质样品进行SDS-PAGE。 </li><li>电子显微镜:细胞核沉淀重悬于含有2％戊二醛在裂解缓冲液从步骤1.6和修复样品在4℃下冲洗样品在锇酸，脱水，并嵌入在环氧树脂中。把70纳米片使用赖克特ULTRACUT超薄切片机。染色醋酸铀和铅和图像使用JEOL 100CX透射电子显微镜的样品。丰富的量化测量完整的细胞核的面积与总面积的材料成像， 我们发现60％至80％的核是完整的。 </li></ol> 重要的考虑因素，富集与净化核。该协议的目的是使心脏染色质蛋白质组分析的研究全球基因调控过程，在疾病的目的。全蛋白质组质谱实验设计的一个重要组成部分，是动态范围和低丰度蛋白能够识别和表征的问题。除提供核染色质特定的生物学信息，丰富的核蛋白质的主要目标，这些低丰度蛋白的检测，增加了调子一样进一步subfractionation的原子核。然而，正如所讨论的，成人的心肌细胞具有致密的细胞骨架成分连接到核膜。我们不认为我们已经完全纯化的细胞核从其他细胞成分。然而，仅通过丰富的原子核，我们有获得编的可接受阈值，使分析的类型描述。 具体而言，我们已经使用了EM，以评估我们的粗核颗粒的纯度，并发现60％至80％的馏分是完整的细胞核中，大约10％的线粒体，其余的碎片。此外，我们知道，从我们以前的研究完整的细胞核人口，而许多肌原纤维丰富的协议（包括微管蛋白和肌动蛋白，采用Western），丰富的某些蛋白质（calsarcin-1），这表明一个真正的生物种群的这些蛋白在心脏核19 此外，我们比较的蛋白质，我们发现本萃取酸染色质馏分中，预测的角色，这些蛋白质通过基因本体。更重要的是，这种基因本体论分析没有考虑到不同的蛋白质的相对丰度（如Western blot检测数据），而是计算所有蛋白质（丰富ed或不）相等时，确定共同的途径和细胞区室。这些途径和细胞区室分析，可以发现在我们以前的出版物中，18,20最重要的是，丰富的酸提取的染色质部分的成功使我们能够识别54个组蛋白变体的存在，在成年小鼠的心肌细胞，18其中许多依赖于一个独特的识别肽，并因此可能不会被检测到没有这个浓缩的协议总心肌细胞蛋白质组的巨大复杂性。我们发现从心核的1048个蛋白质，其中56（5.3％）注释GO分析的一个组成部分的核心利益的核小体（）的一部分。在整个心脏的另一项研究，确定了6180蛋白质，其中只有11个蛋白（0.18％）是注释的一部分的核小体21。这进一步说明了我们的协议实力meaningfuLLY丰富的核蛋白质。 5。蛋白质凝胶和酶蛋白精华蛋白质是由一维SDS-PAGE和胰蛋白酶消化质谱仪上运行分离。 <ol><li>解冻样品在冰上，并确定使用二辛可宁酸（BCA）蛋白质测定每个样品的蛋白质浓度。 </li><li>稀释样品到一个已知浓度的5倍Laemmli缓冲液，煮沸10分钟，并储存在-20°C的一维SDS-PAGE。稀释样品到一个已知的浓度使用尿素提取缓冲液并储存在-20℃下的二维凝胶。 </li><li>加载等于每个泳道中的蛋白量，选择运行凝胶。蛋白质可以被转移到硝酸纤维素膜用于Western印迹法（如在步骤4.1中，与对照）或频带可以削减用于质谱分析，参见以下步骤。 </li><li>使用去离子水，将它转移到一个干净的容器从装置中取出凝胶。覆盖凝胶，黄鹂染色，包装容器铝箔挡光。允许凝胶在振荡器上在室温下孵育至少90分钟。 </li><li>图片黄鹂染色的凝胶（ 图3）UV光，标记的图像乐队将被削减。 我们把每个车道约25毫米波段测量总蛋白质组的研究。 </li><li>将凝胶到一个干净的表面。仅使用材料一直保持密封或喷用乙醇，以防止角蛋白污染。剪下每个波段，然后再切分成3等份。将各带三件一起到自己的标记1.5 ml管。凝胶片可以存储在-20℃，数个月。 </li><li>准备凝胶插头酶消化。月刊凝胶片使用胰蛋白酶于37℃培养过夜。对于详细的凝胶样的协议，请参见我们之前发表的22可以代替胰蛋白酶与胰凝乳蛋白酶消化的低分子量条带，消除胰蛋白酶裂解广泛的组蛋白尾部。 </li></ol> 6。质谱法和数据分析样品上的LC分离，并通过MS / MS进行分析。光谱对蛋白质鉴定蛋白质数据库的搜索。 <ol><li>运行10μl的每个样品通过LC / MS / MS分析。我们使用了纳米流Eskigent LC 75微米反相柱。使用优化的范围内的蛋白质和肽的LC运行。采用一种线性梯度从流动相A（含0.1％甲酸，2％乙腈[ACN]在水中），流动相B:60分钟（0.1％甲酸的乙腈溶液，20％的水）从5％流动相B 50 ％的B，然后从50％B到95％B 15分钟，最后10分钟以恒定的95％B.使用200升/分钟的流速。采集质谱数据，在数据依赖的模式。 我们用一个热的Orbitrap最丰富的母离子的碎片。 </li><li>重复运行至少三个生物，两个技术复制。 （Recommended） </li><li>使用软件（市售的选项包括BioWorks和Xcalibur建立公开可用的选项包括PROWL，X！串联，SpectraST）的UniProt数据库的选择通过光谱对搜索的搜索算法（如SEQUEST或吉祥物）。 </li><li>考虑修改搜索参数，以使半胱氨酸的carbamidomethylation和蛋氨酸氧化，来样加工过程中的两个共同创建的修改。 </li><li>计算使用反向数据库检索的假阳性率。 </li><li>过滤蛋白质的鉴定，只接受比赛的阈值的信心。我们推荐以下的参数来启动:Xcorr 3（+2），4（3），5（+4）; deltaCN&gt; 0.1，共识得分≥20，质量误差2张大为母离子，质量公差0.5沓产物离子，至少2种独特的每蛋白的肽和不超过3错过分裂。 </li></ol> 7。无标记定量 DETErmine的相对丰度蛋白无标记定量（ 图4）。 <ol><li>许多软件程序是公开的或市售的无标记定量质谱数据，包括普查（耶兹的教授组），23的 Elucidator（微软），24 SIEVE（赛默科学），25脚手架（蛋白质组软件）。26这些方案的目的是相互关联的完整肽的质谱信号或两个或两个以上国家的数量与相关蛋白肽测序事件的数量。 </li><li>虽然每个程序采用了类似的分析管道，一些方案限定的类型的具体的分析，可以对数据执行的。最初，从不同的运行中的数据对准，信号强度的归一化和肽峰被选作分析。 </li><li>两种最常见的方法是量化的基础上光谱计数或LC-MS峰面积。丰富比例的计算方法，以确定不同群体之间的肽丰度的变化。 </li><li>这些程序可以与蛋白质组学搜索算法（吉祥物，SEQUEST，X！TANDEM）相关蛋白质鉴定的定量信息。 </li><li>为了确保数据的准确性和可重复性的关键是将两种生物（不同的实验样品）和技术（质谱仪多次运行相同的样品）质谱数据进行复制。 </li><li>和实验条件之间的肽丰度的变化，可以评估由方差分析和绘制通过PCA（ 图5）。 </li></ol> 无标记定量重要的考虑因素。进行无标记定量时，必须特别注意，因为每个样本必须分别进行处理和分析，以确保一致的样品制备，消化时间和LC-MS/MS条件。在对比遇见从不同的运行质谱（标记方法abolic，比较无标记的实验数据的标签，因为缺乏消除他们一起运行的可能性），从而有必要进行高重现性样品分析的各个方面（ 即样品制备， LC和MS步骤）和高的质量准确度质谱仪的使用。 轻微的变化，在一个已知的标准样品制备和分析到账户可以被添加到每个样品的数据归一化，以协助。此外，大多数软件程序允许正常化的信号强度（ 例如，通过调整背景噪声或已知的丰富的分析）后注射，电离和碎裂的差异数据采集帐户。比对算法中存在的大部分的以上提及的软件程序，以协助在纠正在肽的洗脱曲线的差异。利用生物技术复制埃森TiAl金属在这种类型的研究，因为它允许统计分析，以确认蛋白丰度的任何观察到的变化的可重复性和一致性。

<ol>
	<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>

没有利益冲突的声明。

核分离的两个主要方法已被先前评论:27 1是均质冻干组织中的非水溶剂中的贝伦斯技术和第二，其中在这里，我们使用的变形例，在水性的蔗糖/盐溶液均质组织随后由差分或密度梯度离心。 Subfractionation的核酸提取的组织样本，研究与原来的目标是分析组蛋白染色质已自1960年以来，28的重要工具。酸提取协议开发为实现这一目标的纯化核心的核小体的组蛋白?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Dulbeco改良的Eagle培养基</td><td> Invitrogen公司</td><td> 11965 </td></tr><tr><td>蛋白酶颗粒</td><td>罗氏公司</td><td> 04 693 159 001 </td></tr><tr><td> 100微米过滤器</td><td> BD猎鹰</td><td> 352360 </td></tr><tr><td> ULTRACUT超薄切片机</td><td>赖克特</td><td></td></tr><tr><td> 100CX透射电子 显微镜</td><td> ，JEOL美国公司</td><td></td></tr><tr><td>莺</td><td> BioRad公司</td><td> 161-0496 </td></tr><tr><td>组蛋白H2A抗体</td><td>圣克鲁斯</td><td> SC-8648 </td></tr><tr><td> Nucleoporin P62抗体</td><td> BD Biosciences公司</td><td> 610498 </td></tr><tr><td>腺嘌呤核苷酸潮转运抗体</td><td>圣克鲁斯</td><td> SC-9299 </td></tr><tr><td> Bip抗体</td><td>圣克鲁斯</td><td> SC-1050 </td></tr><tr><td>微管蛋白抗体</td><td>西格玛</td><td> T1568 </td></tr><tr><td>组蛋白H3抗体</td><td> Abcam公司</td><td> ab1791 </td></tr><tr><td>核仁纤维蛋白抗体</td><td>细胞信号转导</td><td> C12C3 </td></tr><tr><td> SNRP70抗体</td><td> Abcam公司</td><td> ab51266 </td></tr><tr><td> E2F-1抗体</td><td>赛默飞世尔</td><td> MS-879 </td></tr><tr><td>视网膜母细胞瘤抗体</td><td> BD Biosciences公司</td><td> 554136 </td></tr><tr><td>缺氧诱导因子-1抗体</td><td>诺伟司生物制品</td><td> NB100-469 </td></tr><tr><td> BCA蛋白检测</td><td> Thermo Scientific的</td><td> 23227 </td></tr><tr><td>反相柱</td><td>新的OBJective </td><td> PFC7515-B14-10 </td></tr><tr><td> BioWorks浏览器</td><td> Thermo Scientific的</td><td></td></tr></tbody></table>

quantitative analysis of chromatin proteomes in disease

在位于细胞核内的蛋白质组，其功能是最密切相关的基因调控。成年哺乳动物的心肌细胞的核是独特的，由于双核细胞的百分比很高，1为主的异染色质的DNA的状态，和非分割的性质的心肌细胞呈现成人细胞核在相间的永久状态。2在开发过程中的转录调控和病已经很好的研究在此器官​​，3-5，但仍然相对未开发的核蛋白质负责DNA的包装和表达所扮演的角色，以及如何将这些蛋白质控制转录程序的变化过程中发生的疾病。在发达国家世界，心脏疾病是男性和女性的头号死亡原因。7洞察核蛋白质合作，如何规范这种疾病的进展是至关重要的推进，目前的治疗Ø的选项是。 质谱法是解决这些问题的理想工具，因为它允许一个公正的核蛋白质组的注释和相对定量，这些蛋白质丰富的变化与疾病。虽然有过几次蛋白质组学研究哺乳动物核蛋白质复合物，8-13，直到最近，只有一个心脏的核蛋白质组研究，并认为整个细胞核，而不是研究蛋白质组的在核分舱水平15在很大程度上，这不足的工作是由于隔离心脏核的难度。心肌细胞核内发生的刚性和密集的肌动蛋白-肌球蛋白设备通过它们所连接的多个扩展名从内质网的范围内，心肌细胞的收缩改变他们的整体造型。16此外，心肌细胞线粒体40％第17卷necessita韭细胞核富集除了从其他细胞器。在这里，我们描述了一个协议，用于心肌核浓缩和进一步的分离与生物相关的车厢。此外，我们无标记定量质谱夹层这些馏分技术适合于在各种动物模型和器官系统的代谢标记是不可行的体内实验的详细方法。

图4显示这种形式的相对定量的效用。在左侧面板是个别的单一同位素肽峰（覆盖从不同的鼠标），它已被指定为属于蛋白HMGB1（通过数据库检索标识）。每个峰值，本质上是一个给定的肽的提取离子色谱仪，来自不同的鼠标。这些团体代表三种不同的生理状态:基础，心肌肥厚，心脏衰竭，有三个生物复制为每个组。通过集成的曲线下的面积，可以计算出的相对丰度。在中间面板中的...

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

Quantitative Analysis of Chromatin Proteomes in Disease

允许在质谱高通量分析蛋白的表达和组织了一系列修改。结合亚细胞的分馏和疾病模型，定量质量法和生物信息学在生物系统中发现新的属性。这里描述的方法分析染色质相关蛋白在心脏疾病的设置和容易地适用于其他<em在体内</em&gt;人类疾病的模型。

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

quantitative-analysis-of-chromatin-proteomes-in-disease

Research

JoVE Journal

Medicine

13.0K 次观看.  David Geffen School of Medicine at UCLA. 允许在质谱高通量分析蛋白的表达和组织了一系列修改。结合亚细胞的分馏和疾病模型，定量质量法和生物信息学在生物系统中发现新的属性。这里描述的方法分析染色质相关蛋白在心脏疾病的设置和容易地适用于其他

视频: Quantitative Analysis of Chromatin Proteomes in Disease

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

癌症转移，使用禽流感胚胎模型的定量分析

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

科研

医学

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（q3DISM）

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

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior Cingulate Cortex (PCC) is a potential imaging marker for monitoring the progression of AD. Previous studies did not focus on the functional connectivity between the PCC and nodes in regions outside the DMN, but our study is an effort to explore these overlooked functional connections. For collecting data, we used functional Magnetic Resonance Imaging (fMRI) and Granger Causality Analysis (GCA). fMRI provides a non-invasive method for studying the dynamic interactions between the different brain regions. GCA is a statistical hypothesis test for determining whether one-time series is useful in forecasting another. In simple terms, it is judged by comparing the &#34;Known all the information on the last moment, the distribution of the probability of X at this time&#34; and the &#34;Known all the information on the last moment except Y, the distribution of the probability of X at this time&#34;, to determine whether there is a causal relationship between Y and X. This definition is based on the complete information source and stationary chronological sequence. The main step of this analysis is to use X and Y to establish the regression equation and draw a causal relationship by a hypothetical test. Since GCA can measure causal effects, we used it to investigate the anisotropy of the functional connectivity and explore the hub function of the PCC. Here, we screened 116 participants for MRI scanning, and after preprocessing the data obtained from neuroimaging, we used GCA to derive the causal relationship of each node. Finally, we concluded that the directed connection is significantly different between the Mild Cognitive Impairment (MCI) and AD groups, both from the PCC to the whole brain and from the whole brain to the PCC.

Based on resting-state functional magnetic resonance imaging with Granger causality analysis, we investigated the alterations in the directed functional connectivity between the posterior cingulate cortex and whole brain in patients with Alzheimer&#39;s Disease (AD), patients with Mild Cognitive Impairment (MCI), and healthy controls.

在阿尔茨海默病和轻度认知功能损害的定向功能连接格兰杰因果关系分析中的应用

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

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 patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

体内Menière 病耳和正常听觉的磁共振成像对人颅神经的形态学分析

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

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

解剖性脑 MRI 的全脑分割及变化点分析--Premanifest 亨廷顿病的应用

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