这项工作是由美国国立卫生研究院从赠款（DP2OD007447，R01GM110174和R01AI118891）的资金支持。

1.培养细胞的采集<ol><li>如果细胞悬浮培养，在300 RCF 5分钟收集细胞离心。如果贴壁，吸并丢弃细胞培养基。冲洗附着的细胞用PBS不含Ca 2+和Mg 2+（称为PBS前进）。孵育细胞或胰蛋白酶或胰蛋白酶-EDTA（0.025％ - 取决于细胞系0.5％）有足够的体积，以覆盖板的表面，在37℃直到细胞分离（时间为不同的细胞系而变化）。 </li><li>在300 RCF 5分钟收集细胞，离心。洗多次细胞两个PBS，离心收集。 </li><li>从上标明1.8毫升管或15毫升锥形管刻度大约估计红细胞压积。 注意:从培养的细胞可以管理单元在液氮中冷冻并储存在-80℃下无限期地在这个阶段。 </li></ol> 2.从完整细胞的分离的核</p&gt; <ol><li>解冻的细胞在冰上。 </li><li>解冻核隔离缓冲器（NIB， 表1）。 </li><li>对于每100微升细胞体积制备约5mL NIB缓冲液（ 表1）。对于每1毫升NIB缓冲液，添加蛋白酶抑制剂和稳定剂如下:1微升的1M DTT，2.5微升的200mM AEBSF的，2微升2.5微米微囊和2μl5M的丁酸钠的。与抑制剂NIB从这个角度提出被称为NIB。 注意:如果组蛋白磷酸化进行了研究，包括EDTA无蛋白酶和磷酸酶抑制剂的鸡尾酒。 </li><li>除去NIB缓冲器的第五体积如此制备和添加NP-40的替代（ 表1），以0.2％的最终浓度。其余四个十五体积将被用于洗涤。 </li><li> 1:10细胞沉淀到笔尖洗涤细胞沉淀无NP-40的替代比（体积/体积）。离心在700 RCF 5分钟去除上清液。 </li><li>裂解CEL升通过将它放置在冰上，加入1:10细胞沉淀到笔尖与0.2％的NP-40替代粒料（体积/体积）。 </li><li>如果从组织样品中提取，使用均质迫击炮和杵或DOUNCE均质。培养的细胞可以通过温和移液均化。 </li><li>在冰上孵育5匀浆细胞 - 10分钟。细胞将溶解并释放核。 </li><li>离心机在1000 RCF为5 - 10分钟，4℃。沉淀主要含有细胞核，而上清液主要包含细胞质成分。如果需要保存的胞质级分。 </li><li>通过在1:10（体积/体积）的NIB轻轻重悬它没有NP-40的替代洗细胞核沉淀。 注:此清洗步骤完全是从细胞核中提取组蛋白之前去除洗涤剂的痕迹。 </li><li>离心机在1000 RCF离心5分钟，在4℃并除去上清液。 </li><li>重复步骤2.10 - 2.11至少两次以完全除去NP-40的替代。 NP-40替代的去除是一个明显温柔移液在洗涤步骤不再形成气泡。 </li><li>从组织蛋白提取: <ol><li>冲洗在冰冷的NIB新鲜或冷冻解冻组织。 </li><li>转移组织到培养皿置于与笔尖冰，刚好够保持组织湿润。 </li><li>骰子成最小件（&lt;1毫米）与刀片以增加核隔离的表面接触。 </li><li>转移组织糜到预冷的均化并通过上下吹打在NIB洗净。 </li><li>通过在300 RCF离心5分钟除去缓冲。 </li><li>添加NIB含NP-40的替代的细胞在细胞:缓冲的1:10的比例（体积/体积），并通过5均质 - 10笔。 </li><li>检查细胞裂解，并根据需要重复同质化。一个好的指标，细胞已被裂解为颗粒体积的减少。沉淀应该只包含核。 </li><li>离心在700 RCF 5分钟并保存沉淀。这种粒料可提取1 - 2有更多的时间含NP-40的替代NIB第在1:10（体积/体积）;在这个阶段中，组蛋白被提取出来的染色质和沉淀已经皱缩相当。 </li><li>洗净用2两次 -​​ 3毫升NIB无NP40替代去除洗涤剂的痕迹。 注意:临时停止点:样品可重新悬浮在NIB + 5％甘油的最小体积，并储存在-80℃。 </li></ol></li></ol> 3.从细胞核提取纯化的组蛋白注意:组蛋白是非常丰富的碱性氨基酸残基，使它们能够紧密地与DNA的磷酸骨架相互作用。组蛋白是在细胞核中最基本的蛋白质中，从而使他们能够在冰冷硫酸（0.2 MH 2 SO 4）与来自非组蛋白，其在强酸沉淀污染最小提取。高度浓缩的TCA（33％终浓度）然后可以用于从硫酸沉淀组蛋白酸。 TCA在4℃下储存在棕色瓶中的100％。 <ol><li>悬浮细胞核中1:5（V / V）通过温和移液冷却0.2 MH 2 SO 4（ 表1）。 </li><li>孵育恒定旋转或温和摇动2样品 - 在4℃下4小时。通常情况下，有超过500微升细胞沉淀样品，一个2小时的提取足以提取组蛋白;潜伏期较长，可能会导致其他碱性蛋白质的提取。对于较小的核颗粒（&lt;200微升），4小时提取提供了更好的收益。 </li><li>离心在4℃3400 RCF 5分钟。 </li><li>将上清转移至新管。 </li><li>重复步骤3.3 - 3.4，以消除任何不溶性物质。 </li><li>以沉淀组蛋白，在1的比率冷冻100％TCA（ 表1）添加到所收集的上清液（现在含有组蛋白）:3（体积/体积），以获得33％的最终TCA浓度。通过倒转该管几件T混合输入法。 注:样品会变成混浊在加入三氯乙酸，指示组蛋白的存在。 </li><li>在冰上孵育混合物至少1小时。对于较小的颗粒首发规模，建议一夜沉淀。 </li><li>离心3,400 RCF 5分钟。组蛋白外套管的侧面和也在底部沉积。白色不溶的沉淀也形成在管，它主要包含非组蛋白和其它生物分子的最底部。吸上清，不小心刮边或沉淀。 </li><li>通过使用玻璃巴斯德移液管，冲洗用冰冷的丙酮+ 0.1％的HCl（ 表1）的管，以便覆盖所述沉淀的蛋白质涂覆的侧面和底部。 </li><li>离心3,400 RCF 2分钟，吸上清，不小心刮边或沉淀。 </li><li>采用100％冰冷的丙酮3.10 - 重复步骤3.9。 </li><li>干粒料与空气流或用VACU嗯离心机，或只是离开管开放。丙酮迅速蒸发。 </li><li>溶解与DDH 2 O的（双蒸水）在最小体积可能组蛋白以完全溶解白色层。组蛋白是易溶于水。对于在1.5 ml离心管颗粒，100微升的DDH 2 O通常足以收集组蛋白。 </li><li>离心3,400 RCF 2分钟，将上清转移到新管中。 </li></ol> 4.估计蛋白浓度和纯度的<ol><li>用于测定蛋白质浓度，使用BCA，Bradford蛋白测定法或氨基酸分析（AAA）。不使用通过在280nm的吸光度，作为组蛋白是在芳族氨基酸残差的技术。 </li><li>验证提取的组蛋白通过用15％丙烯酰胺凝胶和考马斯染色（可选）SDS-PAGE分析的纯度。 </li><li>如果纯度高的单组蛋白变体期望，不断的HPLC-UV分馏组蛋白变体（第5）。如果没有，直接看样品制备自下而上的组蛋白PTM分析（第6条）。 </li></ol> 5.通过反相HPLC分离组蛋白变体（可选） 注意:高纯度的组蛋白变体可以通过分馏使用耦合到一个UV检测器的反相HPLC将粗组蛋白混合物来获得。这些纯化的组蛋白对于需要更高的灵敏度和纯度的研究是有用的。然而，对于标准组蛋白PTM表征，该步骤可以被跳过，因为分析是足够灵敏和详尽的。完整的组蛋白变体分馏最好至少需要100 - 300微克原料。 <ol><li>根据原料组蛋白浓度的适当的C 18 5微米柱连接到HPLC:与组蛋白的约100微克，使用2.1毫米×250毫米柱，0.2毫升/分钟的流速;约300微克组蛋白，使用4.6×250毫米柱，0.8毫升/分钟的流速。准备缓冲区A和B使用专用的玻璃器皿如下: <ol><li>准备缓冲液A:5％HPLC级乙腈，0.1％TFA的HPLC级水。 </li><li>制备缓冲液B:95％HPLC级乙腈，0.1％TFA的HPLC级水中。 </li></ol></li><li>列连接到一个UV检测器，并设置吸光度210 - 220纳米。 </li><li>酸化溶解在水中用100％TFA的组蛋白样品达到0.1-1％的TFA的终浓度。 </li><li>平衡用100％缓冲液A中的列以推荐的流率，这相当于大约三个柱体积的至少15分钟。使用该信号来设置UV检测器的零吸光率水平。 </li><li>准备适当大小的管，以手动或自动采样器采集分数。 </li><li>在大约1微克/微升或更高的浓度注入样品。溶解在体积较大的样品可能改变柱杜的平衡环装，导致更低的保留。 </li><li>在1分钟从0至30％的B，在90分钟为30〜60％的B，和60至90％B，在1分钟:运行梯度，编程如下。 </li><li>收集用自动级分收集器在1分钟的间隔分（例如色谱图2中示出）。收集在适当大小管馏分包含整个体积。 </li><li>干下来分级的样品在真空中浓缩。 注意:临时停止点:干组蛋白的级分，可以在室温下保存时间很短（1 - 2天）或在-80℃冷冻长期周期。 </li></ol> 6.组蛋白的化学衍生用丙酸酐为自下而上的分析<ol><li>组蛋白溶解样品中40微升50毫米NH 4 HCO 3，pH值8.0（推荐量:50 - 100微克）。如果样品在纯粹的DDH 2 O，加浓NH 4 HCO 3弥补了50毫米，pH值8。0。 </li><li>润湿P10的枪头到样品用pH试纸没有样品损失，检查pH值。 的 NH 4 OH和甲酸可用于调节pH至8.0。 注意:该协议的以下部分（步骤6.3 - 6.7）应该在最多三个到四个样品的批进行，以保持丙酸酐反应。 </li><li>使用通风橱其中使用丙酸酐随后的步骤。通过在比1混合，用乙腈丙酸酐制备新鲜丙酰化试剂:3（体积/体积）。添加丙酰化试剂在1采样:4（V / V）。对于40微升的组蛋白，加入10微升丙酰化剂。 注:可以在这一步，观察白色碎屑。然而，这主要是含有盐和丙酸，因此没有特别的动作需要服用。 </li><li>快速添加的NH 4 OH以重新建立pH 8.0的溶液中。注意:丙酸酐与肽的游离胺反应产生丙离子的酸性pH值降低。一般，加入的NH 4 OH以使样品与1:5的比例（体积/体积）为宜，以重新建立pH 8.0的; 例如，8微升的NH 4 OH至40微升的样品。 </li><li>涡旋立即混合。 </li><li>检查pH值用相同的方法，步骤6.2。 注意:当pH值大于10.0，具有较高的pKa的其它氨基酸残基的标签是可能的。 </li><li>在室温下孵育样品15分钟。 </li><li>重复步骤6.3 - 6.7，严格执行用于每批次丙酰化试剂的不超过3或4个样品的反应。 </li><li>干的样品下降到10 - 20微升在真空中浓缩。此蒸发的未反应的丙酸酐，乙腈，乙酸和氨气选自NH 4 OH释放。如果样品干燥彻底，不会出现显著样品损失。 注:异丙醇可被用来代替乙腈。然而，乙腈具有较低的表面张力，从而更快速蒸发。 </li><li>与DDH 2 O的悬浮或稀释样品，直到最终体积的40微升的实现。 </li><li>重复步骤6.2 - 6.9。双圆的组蛋白的丙酰化反应，确保完成&gt; 95％。 </li><li>填丙酸酐瓶用氩气，以防止在与该瓶水分接触形成乙酸。 注意:临时停车点:样品可以存储在DDH 2 O的或干的重组-80℃。 </li></ol> 7.蛋白水解消化胰蛋白酶<ol><li>在50毫米NH 4 HCO 3组蛋白悬浮达到1微克/微升以上的最佳浓度。更多稀释样品导致降低胰蛋白酶效率。 注:在此步骤中的组蛋白必须在pH值8.0如果仍然呈酸性，然后加入NH 4 HCO 3盐用吸管尖品尝。 </li><li>在1:10的比例（重量/重量）添加胰蛋白酶到组蛋白样品。 </li><li> Incuba德在37℃，6 - 8小时。 </li><li>在-80℃冷冻停止消化。 </li><li>干倒样品10 - 真空浓缩20微升。 注意:临时停车点:样品可以保存在-80℃。 </li></ol> 8.组蛋白肽在丙酰化N末端注意:本节介绍肽N末端从胰蛋白酶消化所产生的衍生。这种过程改善了最短的肽（ 例如，氨基酸3 - 8组蛋白H3）的HPLC保留，作为丙酰基增加肽的疏水性。 <ol><li>悬浮样品中100毫米NH 4 HCO 3的30微升。 </li><li>重复步骤6.1 - 6.9。 注意:这是正常的样品在真空干燥在此步骤需要较长的时间。 </li><li>悬浮或用50稀释样品-加入100μl的DDH 2 O +或0.1％TFA或0.5％乙酸。注:建议长期储藏醋酸，为TFA facilitatES在长期甲硫氨酸的氧化。另一方面，如果阶段倾翻（第9节）是在同一天进行的，为​​TFA有助于更好色谱保留建议的TFA。 注意:临时停车点:样品可以保存在-80℃。 </li></ol> 9.样品脱盐具有阶段提示注:在此阶段，有存在于样品中的盐。盐妨碍HPLC-MS分析，因为它们电期间电离，抑制来自肽的信号。盐也可以形成对肽离子加合物，降低了对非加成肽的信号强度。作为加成肽将具有不同的质量，该肽将不正确识别或定量。 <ol><li>通过使用P1000枪头，从固相提取盘打孔-C 18材料的圆盘。通过使用熔融石英毛细管推微型盘出P1000尖端和沉积微型盘到P100 / 200枪头的底部。确保对DISK在前端（ 图3）的底部牢固地楔入。 注:P1000顶端具有相当小的孔冲的C盘18。它是适当的切割尖端的最后厘米以具有孔直径较大。 </li><li>使用两个C 18拳在同P100 / P200提示如果脱盐超过25微克的样品。 </li><li>使用离心机适配器持有阶段提示的地方在1.5ml或2 ml离心管中。用慢（300 - 400 RCF）转动;溶剂通常不到一分钟通过树脂。 </li><li>通过用50μl100％乙腈的纺丝激活-C 18材料和消除潜在的污染冲洗树脂。 </li><li>通过慢离心冲洗80微升的0.1％TFA平衡磁盘。 </li><li>酸化样品至pH 4.0或用乙酸低。检查其pH值试纸减少样品损失。负载样本到由慢离心盘。 </li><li>洗通过慢离心80微升的0.1％TFA - 通过冲洗70样品。 </li><li>通过慢离心冲洗70微升75％乙腈和0.5％乙酸洗脱样品。收集在1.5ml管中的样品。 </li><li>在真空中浓缩干燥样品。 注意:临时停车点:样品可以保存在-80℃。 </li></ol> 10组蛋白肽的分析注:NLC-MS平台应被设置为在传统的肽分析完成。建议300 nl流通柱（75微米的ID分析柱，C 18的颗粒），因为它们是灵敏度和稳定性之间的优良折衷-利用200。该MS采集方法可以是有针对性的扫描19或一个与数据无关的采集（DIA）20,21，无论是在代表结果和图4描述的数据依赖性采集（DDA）的组合。 <ol><li>准备HPLC缓冲区 - A:0.1％甲酸HPLC级水; A:在HPLC级乙腈中的0.1％甲酸。 </li><li>编程HPLC方法如下:从0到30％缓冲液B在30分钟内，从30到下一个5分钟100％B，并在8分钟等度100％B。如果HPLC没有编程为样品加载之前自动柱平衡，则包括以下内容:在1分钟梯度从100到0％B等度流动在0％B，10分钟。设置分析至250的流率 - 300升/分钟。 </li><li>程序中的MS采集方法执行两种DDA有针对性的扫描19或20,21 DIA（ 图4）相结合。确保该MS的占空比允许一个全MS扫描每〜2秒，为了有足够的数据点在整个色谱峰，这使更准确的定量。注意:对于C 18层析，平均基线峰宽度为上述的梯度大约30秒。 </li><li>装载大约1微克样品到HPLCÇolumn。 </li><li>按程序运行HPLC-MS / MS方法。 注:在协议中，我们不建议列，MS工具或MS参数细节的细节，因为任何最佳设置，发达国家个人蛋白质组学实验室将适合的方法。蛋白质组学的实验室应利用其优化的设置，由于组蛋白肽单独作为传统的肽。 </li></ol> 11.数据分析<ol><li>导入MS原始文件导入软件来执行峰面积整合。注意:EpiProfile 22被推荐的，因为它是对组蛋白的肽进行了优化;通过使用色谱洗脱的保留时间知识它执行已知组蛋白肽的可靠峰面积萃取。可替代地，天际线23是为目的的另一个理想的软件。 <ol><li>通过在所有的其修饰形式的肽的总面积除以它的面积计算给定的肽的相对丰度。注意:在发现ANALY的情况下，SIS吉祥物建议，以确定修饰的组蛋白肽的光谱。这个工具的性能得到了最近24描述。蛋白质组学所有其它数据库的搜索引擎也可以使用，但在我们的测试中，他们提供较低的性能。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

这里所描述的协议被优化考虑成本，时间和性能。其他制剂也是可能的，但它们有限制，特别是在与MS分析耦合的情况下。例如，高盐提取协议可用于纯化的组蛋白26代替TCA沉淀（第3节）。高盐协议在本质上是温和的，因为它不使用强酸。这保留酸不稳定翻译后修饰，并增加萃取组蛋白的产率，如TCA沉淀共沉淀许多其他染色质结合蛋白。然而，高盐的提取导致含有HPLC-MS / MS过于集中盐样品。通过降低胰蛋白酶温育时间和酶/底物比例27或使用的argc作为消化酶28-30，例如-在另一种制备方法，组蛋白消化可以不丙酰化（8部分6）进行的。但是，建议用衍生丙酸酐，我吨导致更多的疏水性肽，其液相色谱中更好保留的产生。 对于化学衍生，各种有机酸酐进行了评估，其优点全面讨论18。尽管如此，丙酸酐证明效率，最小化的副产物和改进的肽疏水性之间的最佳折衷。潜在地，丙酸酐可以在同位素标记的形式购买;这允许用于复分析由于混合多种样品和在基于来自重标签赋予的不同的质量在MS水平识别它们的可能性。然而，该分析导致的LC-MS色谱的增加的复杂性并减少了可被注入每个单条件的样品量。 在这方面，该协议的一些关键方面应该强调。以下应被用作沟道ecklist找到在执行的情况下，获得阴性结果的程序的错误。首先，核沉淀后沉淀应认真用笔尖不使用NP-40的替代（第2.10），直到（混合过程中明显受到缺乏气泡）完全去除洗涤剂。如果不这样做会损害与酸蛋白提取。二，组蛋白沉淀与TCA（第3.9节）用丙酮沉淀的洗涤之后，是至关重要的。浓酸的存在会损害以下一步，如果直接进行丙酰化和消化（第6.1节）。这将是进行（第5）的情况下，组蛋白分离不成问题。第三，至关重要的是，丙酰化反应迅速进行（第6.3 - 6.7）。要做到这一点，应避免使用相同的丙酰化混合（丙酸酐+乙腈）超过3 - 4次连续采样。此外，pH值是胰酶消化（第7节）的最重要的方面。如果不8.0左右（7.5 - 8.5）消化将是无效的。这可能发生，因为样品将丰富的在这一步丙酸。 NH 4 OH可以加入到必要的。同时，对于熟悉流程的蛋白质组学研究人员就会觉得正常酸化样品终止胰酶消化。这不应该做的，因为它会危及下面的反应， 即肽N末端 ​​（第8.1条）丙酰化。最后，在同样的问题，是要记住用于数据分析该未修饰的肽实际上不是未修饰是很重要的;全部免费赖氨酸残基和N-末端由丙酰化（56.026达）被占用。从而，执行对应唯一的肽序列的质量的提取离子色谱法将导致没有结果。 该方法的局限性是大多与无法检测组合翻译后修饰，由于短肽序列，并在实现真正abun偏见一个变形例的舞蹈，由于这样的事实:在不同的修饰形式的肽可能具有不同的效率电离。第一个问题可以通过这种技术有中间向下或自顶向下的方法（在16中综述）组合来解决。这种类型的分析，即使在技术上更具挑战性，是理想的研究修改共存的频率。此外，它允许组蛋白变体，这是不能总是与自下而上因为一些肽具有在不同组蛋白变体相同的序列实现的更好的歧视。第二个问题，涉及到的离子化效率，可以使用合成肽31的文库来解决。这种方法可以确保翻译后修饰组蛋白的相对丰度的更准确的估计。然而，在大多数实验中，所需的结果是分析条件之间给定修改的相对变化。在这种情况下，这种校正是不必要的，由于这一事实，所有样品都具有相同的岜秒。 总之，这个协议允许组蛋白翻译后修饰的分析，可以在使用NLC耦合到串联MS 3天完成。比MS其他技术，比较即使用如简介讨论了基于抗体的策略，是不适合的，因为它们无法达到甚至接近这个水平的吞吐量。此外，基于抗体的技术不允许新颖改进的发现，但它们仅基于确认和量化预测标记。因此，我们推测组蛋白肽自下而上的蛋白质组学将在明知组蛋白标记，这是在调整的基因表达主角的调控取得蛋白质组学实验室普及，由于直观的优势，从而影响蛋白质的调节。此外，该协议描述的内容包括在样品制备和软件进行数据分析的最新改进，使组蛋白的分析也比较平凡的labora从来没有经历过这种类型的hypermodified肽表征保守党。

表观遗传学定义为在基因表达，通过比改变底层的DNA序列1的其它机制发生遗传改变的研究。表观遗传调控是开发过程中至关重要，因为生物体经受剧烈的表型变化，即使它的DNA含量不发生变化。有适当后生维护所需的几个关键部件，包括组蛋白的翻译后后修饰，组蛋白变体，非编码RNA，DNA甲基化和DNA结合因子，其每一个通过不同的机制2影响基因的表达。例如，虽然DNA甲基化是抑制基因的翻译3，组蛋白变体和组蛋白翻译后修饰是更加动态并且可以以各种方式4影响染色质高度稳定的修饰。 组蛋白翻译后修饰大多局限在N末端尾部，因为它们是最暴露的和灵活的区域的蛋白质。然而，核小体的核心是比较平均的蛋白质5还大量修改。即使组蛋白标记已经被广泛表征在过去十年中，已知组蛋白标记和它们的功能之间的许多环节还不清楚。这主要是由于这样的事实，大部分组蛋白翻译后修饰不与其它翻译后修饰（"串音"）串联单独工作，而是功能变更具体过程，如转录6,7。例如，在基因的p21的组合标记H3S10K14ac激活其转录，它不会与仅在两个翻译后修饰8之一发生。蛋白质HP1契约承认H3K9me2 / ME3和传播修改到附近的核染色质。然而，HP1不能绑定H3K9me2 / 3当相邻S10被磷酸化9。 H3K4的乙酰化抑制结合蛋白spChp1到H3K9me2 / ME3在裂殖酵母 10。此外，该组蛋白赖氨酸ðemethylase PHF8有当3 H3K4me3的翻译后修饰，K9ac和K14ac存在11最高的核小体结合效率。这些例子强调实现组蛋白PTM改变全球概览，而不是集中在单个的修改的重要性。 序列的存在变体也增加了组蛋白分析的复杂性，如组蛋白同种型通常具有高度相似的序列，但往往在染色质不同的角色。例如，H2A.X具有被更容易地在DNA损伤磷酸相比典型H2A 12的C-末端序列，并且需要在雄性小鼠减数分裂13的性染色体的失活;同样，CENP-A则替换着丝粒14规范的组蛋白H3。尽管他们的不同的功能，这些变体共享与各个规范组蛋白的氨基酸序列的一个大的部分，使得它难以识别和分开量化。 </p&gt; 基于抗体的技术，诸如免疫印迹已经被广泛地采用以表征组蛋白。然而，基于抗体的方法是有限的，原因如下:（i）它们只能确认的变形的存在，并且不能识别未知翻译后修饰; （ⅱ）它们被偏压由于共存的标记的存在，其可影响结合亲和力; （ⅲ）它们不能识别组合的标记，因为只有非常少的抗体可用于该目的和（iv）它们高度相似组蛋白变体或类似翻译后修饰（ 例如 ，二-和赖氨酸残基的三甲基化）之间交叉反应。 Egelhofer 等 。描述了商业抗体的超过25％的失败通过斑点印迹或免疫印迹进行的特异性试验，以及特定抗体中的20％以上在染色质免疫沉淀实验15失败。质谱（MS）是目前最合适的分析工具，研究新的和/或组合翻译后修饰，它已经为组蛋白（16审查）被广泛实施。这主要是由于高灵敏度和MS的质量精度，并执行大规模分析的可能性。 自下而上的策略是对组蛋白表征及其翻译后修饰，其中所述完整的蛋白质是酶消化成短肽最常用的基于MS的蛋白质组学策略（5 - 20 AA）。这有利于消化既液相色谱分离和质谱检测。在600的范围群众 - 2000达通常更容易离子化，并具有较高的质量精度和分辨率大于群众识别。 MS / MS碎片也得到了改善，因为短肽通常非常适合碰撞诱导解离（CID）。然而，组蛋白呈现为自下而上的MS是一个挑战，因为它们是在碱性氨基酸残基，即赖氨酸和精氨酸高度富集。因此，胰蛋白酶消化导致过于SM肽的生成所有LC保留和翻译后修饰的明确定位。为了规避这个问题，我们的协议包括赖氨酸和肽N端化学衍生17。建议使用丙酸酐为有效的化学衍生，相对于其他的试剂18。这样衍生块未修饰的和单甲基赖氨酸残基的ɛ氨基团，使胰蛋白酶仅在精氨酸残基的C末端执行蛋白水解。衍生的胺不能与溶液交换质子并因此将肽一般只有双或三电荷，便利MS和MS / MS检测。此外，N-末端衍生增加肽的疏水性，因此反相色谱保留。这里，我们描述了工作流程，纯化组蛋白和通过自下而上蛋白质组学（ 图1），用于PTM分析做好准备。这一策略实现了单组蛋白标记和组合商标的F量化或组蛋白翻译后修饰是在氨基酸​​序列比较接近。

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Trypsin 0.25% EDTA</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:151px;">25200056</td>
			<td style="width:217px;">For harvesting cells</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">PBS</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:151px;">14200075</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Tris</td>
			<td style="width:157px;">Roche</td>
			<td style="width:151px;">77-86-1</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Potassium Chloride</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">BP366-500</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sodium Chloride</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">S9888</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Magnesium Chloride hexahydrate</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">M9272</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Calcium Chloride, anhydrous</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">C1016</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sucrose</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">BP220-1</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">DTT</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:151px;">15508-013</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">AEBSF</td>
			<td style="width:157px;">EMD Millipore Corp</td>
			<td style="width:151px;">101500</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Microcystin</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">M4194</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sodium Butyrate</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">B5887</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X)&#160;</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">78445</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">NP-40 Alternative</td>
			<td style="width:157px;">CALBIOCHEM</td>
			<td style="width:151px;">492016</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sulfuric Acid, ACS grade</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:151px;">7664-93-9</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Trichloroacetic acid</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">T6399</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Acetone</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">179124</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">HCl</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:151px;">A144-500</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Bradford reagent</td>
			<td style="width:157px;">Biorad</td>
			<td style="width:151px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">30% acrylamide/bis 29:1 -- 500ml</td>
			<td style="width:157px;">Biorad</td>
			<td style="width:151px;">1610156</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Coomassie</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">20278</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">C18 Column (5um) 2.1mm x 250mm</td>
			<td style="width:157px;">Grace</td>
			<td style="width:151px;">218TP52</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">C18 Column (5um) 4.6mm x 250mm</td>
			<td style="width:157px;">Grace</td>
			<td style="width:151px;">218TP54</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">HPLC grade acetonitrile</td>
			<td style="width:157px;">Fisher Chemical</td>
			<td style="width:151px;">A955-4</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">HPLC grade water</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">W6 4</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">TFA</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:151px;">A11650</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Ammonium Bicarbonate</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">A6141</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">ammonium hydroxide</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">338818</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">propionic anhydride</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">240311</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Sequencing grade modified trypsin</td>
			<td style="width:157px;">Promega</td>
			<td style="width:151px;">PRV5113</td>
			<td>For digesting histones for MS</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Acetic Acid</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">49199</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">C18 extraction disk</td>
			<td style="width:157px;">Empore</td>
			<td style="width:151px;">2215</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:432px;">Formic Acid</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:151px;">F0507</td>
			<td></td>
		</tr>
	</tbody>
</table>

complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: from histone extraction to data analysis

核小体是染色质的最小结构单元，围绕组蛋白八聚体包裹的DNA的147个碱基对组成。组蛋白功能是由广泛的翻译后修饰通过核蛋白无数介导的。因为它们调节涉及基因调控，DNA修复和染色体凝集染色质结构和招募的酶，这些修改是对核完整性至关重要。尽管科学界的一个大的部分采用的基于抗体的技术来表征组蛋白PTM丰度，这些方法是低通量和针对hypermodified蛋白质偏置，如该表位可能被附近的变形受到阻碍。这个协议描述了使用纳米液相色谱（NLC）和质谱（MS），用于组蛋白修饰的准确定量的。 S内此方法被设计成表征了大量的各种组蛋白翻译后修饰的和几组蛋白的相对丰度变英格尔分析。在这个协议中，组蛋白衍生与丙酸酐接着消化胰蛋白酶，以产生5的肽 - 长度为20个氨基酸。消化后，将组蛋白肽的新暴露N-末端衍生化NLC-MS期间改善色谱保留。该方法允许为组蛋白翻译后修饰跨越四个数量级的相对定量。

作为一个例子，我们分析了来自人胚胎干细胞（胚胎干细胞）有和没有视黄酸（RA）的刺激，开始用200μl细胞沉淀中提取的组蛋白。在细胞培养中的RA的存在导致胚胎干细胞分化。从细胞沉淀中，约50 - 100微克的组蛋白的提取，这是比足以执行组蛋白肽的多个LC-MS注射更多。衍生，消化和脱盐后，将样品装载到75微米×15cm的-C 18以串行模式柱（粒径3微米，孔径300埃）用耦合到微流体芯片高效液相纳米色谱系统混合线性阱四极杆 - 轨道阱质谱仪。使用DIA进行MS采集。 （数据未示出）平行，将样品进行了分析，使用耦合到混合离子陷阱的Orbitrap质谱仪的纳米流UHPLC一个DDA方法。在每个周期，用290扫描范围进行到1400 米/ Z，60000（200 M / Z）和AGC 10的分辨率6，然后，数据依赖性获取模式用动态施加一个完整的MS轨道阱检测排除30秒。 MS / MS扫描随访从最剧烈的母离子。带之一的电荷状态的离子被排除的MS / MS。用2 米/ z与隔离窗口。离子使用碰撞诱导解离（CID）以35％的碰撞能量分段。离子阱检测与正常扫描范围模式和正常扫描速度为10 4 AGC使用。 原始MS数据分析采用软件对前体离子和碎片离子色谱图的提取，即地平线23和EpiProfile 22。 EpiProfile已经为组蛋白肽进行了优化，因为它集成了智能峰面积的提取由于赛前的以前的知识潮保留时间。另一方面，天际线为DIA分析进行了优化，并因此显示DIA图（ 图4和5A）是从该软件的屏幕截图。从所提取的离子色谱图，所述曲线下的面积被检索，这是用于估计各肽的丰度。计算的[M + H]的色谱峰的面积+，[M + 2H] 2+，[M + 3H]相同肽3+离子，尽管在大多数情况下，[M + 2H] 2+是普遍形式。这提供了肽的给定修改形式的原始丰盈。为了实现翻译后修饰的相对丰度，的一组蛋白肽的所有不同修饰形式的总和被认为是100％，并且特别肽的面积通过的总面积在所有的其修饰形式划分为组蛋白肽。 组蛋白肽存在的VA的等压形式riety（ 图5）。同量异序肽， 例如，K18ac和K23ac，只能在MS / MS级，其中，他们的独特的碎片离子被用于确定所述等压物种（ 图5A和5B）的比例进行定量。此比率被用来划分在两个物种之间的色谱峰的面积。当使用DDA，这些等压形式被包括在目标块的列表，因为这些肽需要通过其整个洗脱被选择用于碎片，这不会在标准DDA实验发生。同量异序物质的相对丰度的鉴别，然后通过监测碎片离子的洗脱曲线进行。在另一方面，DIA采集类型不需要任何包含列表。然而，这种类型的获取方法是不与传统的数据库检索兼容，因而可能防止未知修饰的肽的发现。 <p cl屁股="jove_content"FO:保together.within页="1"&gt;赖氨酸乙酰化（+ 42.011道尔顿）通过使用高分辨率MS采集（&gt; 30,000）从近等压三甲基化（+ 42.047达）歧视。此外，乙酰化比三甲基更加疏水，从而导致乙酰肽晚于各自三甲基那些洗脱。相同的肽的未经修饰的形式洗脱甚至以后，由于这样的事实，该赖氨酸丙酰化。总之，疏水性的，用于与一个可修改站点的肽的顺序是二 - 和三甲基化&lt;乙酰&lt;未修改（丙酰化）&lt;单甲基（丙酰化）。 刺激分化（ 图6A和6B）时的hESC表现出明显的减少乙酰肽。这并不奇怪，因为以前的结果在胚胎干细胞相比，分化的人25日报道更高乙酰化，反映了多能性染色质的普遍宽容的本性。通过专注于组蛋白H3，35个不同的修饰形式进行定量（ 图6C）。然而，可以使用这种方法进行调查，所有的组蛋白proteoforms超过200个，包括所有的组蛋白变体和低丰度修改（数据未显示）。此外，我们的分析表明，可以技术重复之间获得高的再现性，由误差条（表示±标准偏差）的小尺寸所证明。综上所述，本节描述了如何提取使用NLC-MS数据组蛋白修饰肽的相对丰度。 <img alt="图1" src="/files/ftp_upload/54112/54112fig1.jpg" /> 图 1: 工作流程自下而上的MS / MS组蛋白分析中所示的十个步骤为组蛋白分析，包括的每个步骤所需的时间的估计。<html>部分号在括号中给出作为本中的手稿。第5节，描述样品分级分离的各种组蛋白变体，可以省略，除非有必要为给定的变体的高度敏感的分析。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54112/54112fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54112/54112fig2.jpg" /> 图2: 反相液相色谱流动组蛋白变体分馏和考凝胶 （A）代表完整的组蛋白分离LC-UV色谱。组蛋白H3变体可从彼此根据其洗脱时间来区别。馏份可以手动或使用自动级分收集器收集。（B）的组蛋白纯化的三次重复考马斯凝胶。="htt​​ps://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54112/54112fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54112/54112fig3.jpg" /> 图3:阶段倾翻插头 P1000的枪头， 制作 ，冲床从固相萃取盘（第二盘）由-C 18材料的磁盘。在微型盘会粘在尖端（中图），以便它可以被推出到使用任何种类的小毛细管的一个较小的P100 / 200枪头。在这个例子中，我们使用了一个700微米的外直径的熔融石英管。微型盘要推到P100 / 200枪头，直到不能再往前走（最后一个面板）的底部。舞台提示准备组蛋白脱盐，因为它有足够的能力来保留足够的样本材料为众多的重复。特别是，一个微型盘就足够了15 - 20微克Sa的mple。如果需要更多的样本，多个磁盘可以装在另一个。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54112/54112fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54112/54112fig4.jpg" /> 图4:DDA和DIA方法的示意图当使用DDA，该MS扫描周期是根据它们的强度和电荷状态特征在于前体离子的MS / MS碎裂的顺序选择。一旦前体离子已经支离破碎它被放置到排除列表，以避免相同的肽的重复选择，以便MS可以"挖"到不太丰富的信号。本次收购的方法是选择在蛋白质组学发现模式的技术。量化是通过给定离子的旁边所识别的MS全扫描信号进行积分来实现/MS谱。在DIA，整个m / z范围在每一个扫描周期支离破碎。该方法是不太适合发现模式，但它产生的所有离子，前体和产物的色谱图。这将导致更自信量化和等压形式的歧视。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54112/54112fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54112/54112fig5.jpg" /> 图5: 等压肽量化 （A）的 2等压肽组蛋白的分析通常是丰富的实例。提取的离子色谱的前体质量和相对同位素（上图）（XIC）是相同的。然而，产物离子的XIC（下）允许两个同量异序形式歧视。值得注意的是，只有唯一的碎片离子应该是我们ED估计这两个物种的相对丰度。独特的碎片离子两个描述肽（B）表示（以红色突出显示），在具有至少一个等压相当于智人常用分析肽（C）列出。序列变异的组蛋白上市肽表示。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54112/54112fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54112/54112fig6.jpg" /> 图6: 使用和不使用维甲酸治疗人类胚胎干细胞的代表性结果 （A）的组蛋白H3肽KQLATKAAR的相对定量-在其所有的修改proteoforms的（AA 18 26）。相对丰度使用所有proteoforms为100％（相对的估计未示出的未修饰肽的ative百分比）（B）中的组蛋白H3肽KSTGGKAPR（AA 9的相对定量。 - 17）（℃）与和不与视黄酸细胞治疗规范组蛋白H3检测肽的相对丰度。该图表示其中的两种治疗给定的修改是更丰富（&gt; 50％）。总体而言，我们证明了组蛋白H3乙酰化后，大部分细胞分化诱导的赖氨酸残基的降低。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54112/54112fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 解决方案＃ </td><td colspan="7"> 组成 </td></tr><tr><td> 1 </td><td colspan="7">核分离缓冲液（NIB）股制备如下并冷冻贮存于-20℃，C 100毫升分装;解冻NIB可以储存在4℃几周:15毫摩尔Tris，60mM的氯化钾，15 mM氯化钠，5mM的MgCl 2的，1毫米氯化钙2，和250mM的蔗糖。缓冲液的pH调节至7.5，用HCl。 </td></tr><tr><td> 2 </td><td colspan="7">蛋白酶抑制剂（添加新鲜的缓冲区使用前）:1米二硫苏糖醇（DTT）在DDH 2 O（1000倍）; 200毫米AEBSF在DDH 2 O（400X） </td></tr><tr><td> 3 </td><td colspan="7">磷酸酶抑制剂（添加新鲜到缓冲器使用前）:2.5μM微囊在100％乙醇（500倍） </td></tr><tr><td> 4 </td><td colspan="7"> HDAC抑制剂（补充新鲜到使用前的缓冲区）:5丁酸钠，通过用NaOH至pH7.0（500X）5米丁酸作出滴定</td></tr><tr><td>五</td><td colspan="7"> NP-40备选:10％V /在DDH 2 o的钒</td></tr><tr><td> 6 </td><td colspan="7"> 0.2 MH 2 SO 4在DDH 2 O </td></tr><tr><td> 7</td><td colspan="7">三氯乙酸（TCA）:100％w / v的在DDH 2 O </td></tr><tr><td> 8 </td><td colspan="7">丙酮+ 0.1％盐酸（HCl）的0.1％体积/体积盐酸的丙酮</td></tr></tbody></table> 表1.解决方案。

Watch this Scientific Journal Video about Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis at JoVE

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法:从组蛋白提取数据分析

这个协议概述了表征使用质谱（MS）组蛋白的翻译后修饰的完全集成的工作流程。该工作流包括使用纳米流液相色谱法和用于数据分析的指令的细胞培养物或组织中，组蛋白衍生和消化，MS分析组蛋白纯化。该协议被设计为内完成2 - 3天。

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

complete-workflow-for-analysis-histone-post-translational

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

28.8K 次观看.  University of Pennsylvania. 这个协议概述了表征使用质谱（MS）组蛋白的翻译后修饰的完全集成的工作流程。该工作流包括使用纳米流液相色谱法和用于数据分析的指令的细胞培养物或组织中，组蛋白衍生和消化，MS分析组蛋白纯化。该协议被设计为内完成2 - 3天。 

视频: Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

科研

生物学

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

检测组蛋白修饰在植物叶片

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

哺乳动物链接组蛋白亚型表达分析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

自下而上和猎枪蛋白质组学鉴定一个全面的蛋白质组耳蜗

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...