Name: adaptation of hybridization capture of chromatin-associated proteins for proteomics to mammalian cells
Uploaded: 2018-06-01T13:30:00.000+00:00
Duration: 9 min 27 s
Description: Watch this Scientific Journal Video about Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells at JoVE.com

这项工作得到了 NIH 资助 P50HG004952 和 R01GM109099 的支持。

1. 捕获寡核苷酸设计
<ol><li>
设计在目标区域/s 的杂交捕获期间使用的寡核苷酸小组。
	<ol>
<li>目的设计每个目标区域的4–8寡核苷酸, 但最低限度, 设计至少一个针对目标区域的每一端的寡核苷酸。</li>
		<li>如果目标序列是长的 (> 500 基对), 设计出尽可能分散的寡核苷酸, 以确保在整个目标区域有效地丰富染色质。 注意: 我们观察到了4–8寡核苷酸的最佳捕获。虽然这个过程可以很好地与更多的寡核苷酸, 观察在大量寡核苷酸被使用时的富集率下降是罕见的。</li>
	</ol>
</li>
	<li>设计类似熔融温度的寡核苷酸, 确保它们不形成强发夹, 也不具有较强的交互作用 (考虑到杂交将在42ᵒC 进行)。 注: 如果选择了立足点洗脱 (4.12.3), 则必须将额外的外部8基序列添加到寡核苷酸中, 并且在洗脱17时需要互补的 "释放" 寡核苷酸。一些商业软件工具可以用来选择和分析寡核苷酸。25的市场汇率已经显示出良好的效果, 但根据基因组的大小和序列, 捕获寡核苷酸可以包含在20–80基地之间的某处。通常, 捕获寡核苷酸的熔化温度接近62ᵒC, 但这可能会改变, 取决于它们的长度。一致的熔化温度和/或寡核苷酸长度在整个捕获组合可以帮助避免不必要的偏见, 在浓缩。</li>
	<li>设计捕获寡核苷酸与生物素基团 (最好是分离的间隔从捕获序列) 在 3 ' 年底。</li>
</ol>2. 细胞培养
<ol><li>
生长人类永生细胞 (如此处显示的 GM12878) 或其他细胞系在 RPMI 1640 培养基中补充1% 青霉素-链霉素, 2 毫米 l-谷氨酰胺和5% 胎牛血清 (血清), 37 ᵒC 和 5% CO2。
	<ol>
<li>种子初始冷冻整除数 (2–5 x 10 5 细胞) 在 T-25 瓶中, 6 毫升 RPMI 1640 介质在2.1 中制备.</li>
		<li>监测细胞密度和活力。<ol>
<li>取一个具有代表性的细胞整除, 用合适的染料如台盼蓝染色。</li>
			<li>使用 hemocytometer 或自动单元格计数器测量细胞密度。</li>
		</ol>
</li>
		<li>在细胞达到 1 x 106细胞/mL 密度之前, 将细胞向下旋转 100 x g 以达到5分钟, 并将细胞转移到一个 T-75 烧瓶中, 25 毫升的 RPMI 1640 介质在2.1 中制备。将细胞生长直到细胞密度接近 1 x 106细胞/mL。</li>
		<li>将细胞转移到一个 T-150 烧瓶, 在38毫升的 RPMI 1640 培养基 (2.1), 并增加两天的细胞。 注: 永生细胞在悬浮液中生长。生长永生细胞的一般准则建议在 0.2–1 x 106细胞/mL 之间保持细胞密度。由于这项技术所需的大量材料, 本议定书的目的是将细胞生长到细胞密度之外。细胞生长协议可以由读者修改和修改, 具体取决于要使用的单元类型。</li>
		<li>以 100 x g 为5分钟旋转单元格, 并用重悬10毫升 RPMI 1640 介质 (2.1) 中的细胞, 然后将悬浮液倒入 850厘米2 滚轮瓶中, 其中包含500毫升的介质。孵化的细胞在相同的条件下, 但在这一点上开始使用恒定旋转 (~ 0.5 rpm)。</li>
		<li>监视细胞生长并在需要时改变介质 (通常每3–4天)。让细胞生长到密度尽可能接近 2 x 106单元格/mL (1 x 109单元格)。</li>
		<li>一旦达到所需的细胞计数, 通过将细胞向下旋转 100 x g 以5分钟并用重悬细胞, 并在36毫升的 RPMI 1640 培养基 (2.1) 中对细胞进行采集。将细胞悬浮液转移到50毫升锥形管上。用小整除数进行细胞计数和 DNA 提取。</li>
	</ol>
</li>
	<li>
交联通过加入1毫升37% 甲醛达到最后浓度的1%。 注意: 甲醛是一种致癌物, 应在通风罩中处理。<ol>
<li>在室温下 (RT), 用旋转 (~ 30–60 rpm) 孵化细胞10分钟。</li>
		<li>通过加入2毫升的2.5 米甘氨酸溶液来淬火交联反应, 最终浓度为125毫米。</li>
		<li>用旋转 (~ 30–60 rpm) 在 RT 上孵化细胞5分钟. 通过离心 100 x g 为5分钟, 用冰冷 1x PBS 清洗细胞两次。</li>
	</ol>
</li>
	<li>继续下一步或并用重悬颗粒在5毫升细胞裂解缓冲 (5 毫米 Hepes, 85 毫米氯化钾, 0.5% Igepal, 蛋白酶抑制剂 (PI)) 和抑制冻结的悬浮液滴下降的液氮。将样品存放在-80 ᵒC。 注: 准备 > 25 毫升的细胞裂解缓冲液没有 Igepal 和 PI, 并添加这些试剂新鲜时, 准备使用。一旦添加了 Igepal 和 PI, 避免了细胞溶解缓冲液的长期贮存。在4ᵒC 储存细胞裂解缓冲液长达3月。Igepal 是一种非离子的无变性洗涤剂。NP-40 可以作为 Igepal 的替代品, 但我们没有在这方面使用它, 因为当样品最终通过质谱分析来表征 DNA 结合蛋白时, 我们并没有用到 NP-40。</li>
</ol>3. 裂解和剪切
<ol><li>制备 > 15 毫升核裂解缓冲液 (10 毫米 EDTA, 50 毫米三 pH 8.0, 1% SDS, PI)。 注: 添加 PI 新鲜, 只是要使用的金额。一旦加入 PI, 避免了细胞核溶解缓冲的长期储存。核裂解缓冲液可储存在 RT 上长达1月。</li>
	<li>并用重悬20毫升细胞裂解缓冲液中的颗粒 (含 Igepal 和 PI)。让小球融化在冰上, 混合好一次解冻。让样品坐在冰上再加10分钟。离心细胞在 400 x g 在4ᵒC 5 分钟。</li>
	<li>放弃上清和并用重悬6毫升核裂解缓冲液中的细胞 (如果细胞不形成悬浮体, 体积可以增加)。</li>
	<li>
在准备超声波时, 将样品均匀地分在 ~ 6–8离心管 (每管600到1000µL)。
	<ol>
<li>将样品放在冰上或冷的架子上, 用 microtip 的 sonicator 油脂实验样品。使用65% 振幅与 5 x 二十年代恒定的爆发, 允许暂停降温至少四十年代之间的脉冲。 注: 如果体积增加 > 每样品10毫升, 可改用玻璃冷却电池。这时需要越来越多的爆裂。Sonicators 差别很大。条件可能必须由读者优化。其他超声波的替代品也可以使用。</li>
	</ol>
</li>
	<li>离心细胞在 1.2万 x g 10 分钟在4ᵒC, 转移上清到新的管, 并丢弃小球。</li>
	<li>使用荧光法估计总回收率。</li>
	<li>继续执行下一步或快照冻结, 并将该示例存储在-80 ᵒC。</li>
	<li>可选: 采取整除, 逆转 crosslinks 通过孵化它隔夜在67ᵒC 在0.3 米氯化钠。清除 DNA 并在 Bioanalyzer 中运行以确定片段长度。大部分的碎片应该在500和 1000 bp 之间。 注意: 此时停止并将样品存储在-80 ᵒC 是可以的。</li>
</ol>4. 杂交捕获
<ol><li>准备500毫升2x 杂交 (Hyb) 缓冲 (200 毫米 MES, 2M NaCl, 40 毫米 EDTA, 0.02% Tween-20) 和500毫升的洗涤缓冲器 (200 毫米氯化钠, 0.2% SDS, 50 毫米三 pH 值 8)。存储 Hyb 和洗涤缓冲器在4ᵒC 和室温, 分别为3月。使用 2x Hyb 缓冲区的一半来准备1x 杂交缓冲。还存储在4ᵒC 3 月。 注: 在上一步中, 杂交捕获的总体积将是结果样品的体积的两倍 (如前所述, 样品体积约为6毫升, 因此总杂交量为12毫升)。</li>
	<li>预留珠等于总杂交捕获量的 5% (在本例中为600µL)。在下面的步骤中, 需要一个合适的和合适的磁铁来处理珠子。</li>
	<li>用两次珠量 (1200 µL), 用 1x Hyb 缓冲器清洗珠子三次。并用重悬1200µL 2x Hyb 缓冲器中的珠子。将管放在磁铁上, 直到解决方案清除并卸下缓冲区。</li>
	<li>在1.5 毫升离心管中进行预清反应, 每管最大700µL 样品。添加足够的珠子 (5%) 和 2x Hyb 缓冲器稀释 Hyb 缓冲到 1x (10 管600µL 的样品, 120 µL 洗涤珠悬浮在 2x Hyb 缓冲器, 480 µL 的 2x Hyb 缓冲区)。孵化他们为10分钟在31ᵒC 与末端结束自转 (~ 30–60 rpm)。</li>
	<li>把管子放在磁铁上, 直到珠子固定并将样品转移到新的管子上。扔掉珠子。 注: 从这一点上, 建议低结合管。</li>
	<li>并用重悬捕获寡核苷酸到一个工作解决方案的 10 pmol/µL, 并添加4µL 这个解决方案, 每管。 注意: 不同区域可以相互控制, 但建议将捕获寡核苷酸包含在其序列上, 以作为一个真正的负控制。</li>
	<li>孵化管40分钟, 在42ᵒC 与端结束旋转 (~ 30–60 rpm)。</li>
	<li>在孵化期间, 预留所需的珠子 (0.3 µL/pmol 捕获寡核苷酸)。按照步骤4.3 中描述的三次清洗珠子, 但并用重悬 1 x Hyb 缓冲区中的单元格。</li>
	<li>将洗涤过的珠子添加到样品中, 并在 RT 上孵化出30分钟的样品以结束结束旋转 (~ 30–60 rpm)。把管子放在磁铁上, 一旦珠子固定了, 就把上面的清掉。</li>
	<li>
洗珠子
	<ol>
<li>添加1.2 毫升的洗涤缓冲和孵化的样本5分钟与结束结束旋转 (〜 30–60 rpm) 在 RT. 把管子放在磁铁上, 等几分钟, 直到溶液清除。删除缓冲区并重复此步骤。</li>
		<li>添加1.2 毫升的洗涤缓冲和孵化它为1小时与结束结束旋转 (~ 30-60 rpm) 在 RT. 把管子放在磁铁上, 等几分钟, 直到溶液清除。卸下缓冲区, 然后重复此步骤, 将潜伏期缩短到第二次30分钟。</li>
		<li>增加200µL 的洗涤缓冲和孵化它为15分钟以结束结束自转 (~ 30–60 rpm) 在31ᵒC。把管子放在磁铁上, 等几分钟, 直到溶液清除。删除缓冲区。</li>
		<li>添加200µL 的 PBS 和孵化它为5分钟与结束结束旋转 (~ 30–60 rpm) 在 RT. 把管子放在磁铁上, 等几分钟, 直到溶液清除。删除缓冲区并重复此步骤。</li>
	</ol>
</li>
	<li>
简单洗脱
	<ol>
<li>添加40µL 的分子级水的珠子和孵化它在94ᵒC 5 分钟。</li>
		<li>把管子放在磁铁上, 等几分钟, 直到溶液清除。</li>
		<li>把上清液转移到新的管子里。扔掉珠子。</li>
		<li>进行蛋白质组分析或将样品存储在-80 ᵒC。 注: 这种类型的洗脱工作很好, 为以后的 qPCR 分析和一些蛋白质组学测量, 但在其他情况下, 两种替代方法所描述的产量 "清洁" eluates。</li>
	</ol>
</li>
	<li>
DNase 洗脱。
	<ol>
<li>添加40µL 1x DNase 缓冲器和 2 U 的 DNase 每管。</li>
		<li>在37ᵒC 孵化试管15分钟。</li>
		<li>把管子放在磁铁上, 等几分钟, 直到溶液清除。</li>
		<li>从珠子中取出洗脱样品, 将样品放在新的低结合管中。</li>
		<li>将试管孵化94ᵒC 7 分钟, 使酶失效, 并进行蛋白质组分析。 注: 如果观察到对蛋白质组分析的干扰, 可以使用较低的 DNase 浓度。如果使用这种洗脱方法, DNase 处理完全破坏样品中的 DNA 材料, 就不可能计算 qPCR 的捕获充实。</li>
		<li>进行蛋白质组分析或将样品存储在-80 ᵒC。</li>
	</ol>
</li>
	<li>
立足点洗脱17。 注: 此方法不推荐用于较长的寡核苷酸 (> 40 碱基)。这种方法要求捕获寡核苷酸包含额外的8碱基, 不与基因组序列和互补释放寡核苷酸重叠, 以取代捕获寡核苷酸的目标。<ol>
<li>稀释 2 nanomoles 释放寡核苷酸在40µL 的 SSC 缓冲。</li>
		<li>在 RT 中孵化20分钟。</li>
		<li>把管子放在磁铁上, 等几分钟, 直到溶液清除。</li>
		<li>从珠子上取出溶液。</li>
		<li>进行蛋白质组分析或将样品存储在-80 ᵒC。</li>
	</ol>
</li>
</ol>5. qPCR 与引物对目标捕获区的俘获率的评价
<ol><li>使用适当的软件或在线工具设计目标区域的引物和探头。 注意: 在本实验中, DUSP3和DUSP5的启动子区域是目标对象。所用的引物和探头如下。DUSP3: 底漆 1-GTG TTT AGG TTC CCT, 底漆 2-CGG, TGC 铁通 CG, 家族标记探针-ATG CCC GAA。DUSP5: 底漆 1-热重 GAA AGC CCT GTG TC, 底漆 2-海湾合作委员会的 AAC CCC AAA AG, 家族标记探针 TGC 标签 agc agc, ATG AGG TT。</li>
	<li>取一个整除的洗脱液 (不超过10µL), 并稀释1:10 与分子级H2 O。</li>
	<li>提取步骤2.1.7 中的一个整除数的 dna, 并使用该 dna 样本通过在分子级 H 2 O 中做五系列1:10 稀释来制作标准曲线.</li>
	<li>准备一个 qPCR 主混合 (各种商业替代品), 添加底漆和探针, 并分配适当的数量 (15 µL, 如果运行一个20µL 反应) 在每个井。</li>
	<li>添加5µL 的两个: a) 稀释样品, b) 标准曲线或 c) 分子级 H2O (作为没有模板控制) 每个井。</li>
	<li>密封板和离心机在 100 x g 1 分钟。</li>
	<li>在具有以下参数的 qPCR 系统中运行: 5 分钟在95ᵒC 其次40周期95ᵒC 二十年代, 59 ᵒC 二十年代和72ᵒC 为二十五年代。</li>
	<li>使用标准曲线的驴产量和炒捕获, 以评估捕获特异性。</li>
</ol>

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I., 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=Chromatin+proteins+captured+by+ChIP-mass+spectrometry+are+linked+to+dosage+compensation+in+Drosophila.">Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila.</a> Nat Struct Mol Biol. 20 (2), 202-209 (2013).</li><li>Byrum, S. D., Raman, A., Taverna, S. D., Tackett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChAP-MS:+A+Method+for+Identification+of+Proteins+and+Histone+Posttranslational+Modifications+at+a+Single+Genomic+Locus.">ChAP-MS: A Method for Identification of Proteins and Histone Posttranslational Modifications at a Single Genomic Locus.</a> Cell Rep. 2 (1), 198-205 (2012).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+immunoprecipitation+(+eChIP+)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated immunoprecipitation ( eChIP ) using CRISPR.</a> Biochem. Bioph. Res. Co. 439 (1), 132-136 (2013).</li><li>Liu, X., 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=In+Situ+Capture+of+Chromatin+Interactions+by+Biotinylated+dCas9.">In Situ Capture of Chromatin Interactions by Biotinylated dCas9.</a> Cell. 170 (5), 1028-1043 (2017).</li><li>Myers, S. A., Wright, J., Zhang, F., Carr, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-APEX-mediated+proximity+labeling+enables+discovery+of+proteins+associated+with+a+predefined+genomic+locus+in+living+cells.">CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells.</a> bioRxiv. , (2017).</li><li>D&#233;jardin, 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 (1), 175-186 (2009).</li><li>Buxton, K. E., 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=Elucidating+Protein-DNA+Interactions+in+Human+Alphoid+Chromatin+via+Hybridization+Capture+and+Mass+Spectrometry.">Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry.</a> J. Proteome Res. 16 (9), 3433-3442 (2017).</li><li>Kennedy-Darling, J., Holden, M. T., Shortreed, M. R., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+programmable+release+of+captured+DNA.">Multiplexed programmable release of captured DNA.</a> Chembiochem. 15 (16), 2353-2356 (2014).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+specific+genomic+regions+and+identification+of+associated+molecules+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Chromatin Protoc. Third Ed. , 43-52 (2015).</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 (4), 870-882 (2009).</li></ol>

没有任何利益冲突可供披露。

这里描述的 HyCCAPP 方法有许多独特的特点, 使它成为一个强有力的方法来揭示 DNA-相互作用, 否则将仍然难以捉摸。这个过程的性质使 HyCCAPP 在基因组的不同有机体和区域工作的灵活性。然而, 这是一个方法, 有几个限制要考虑。
HyCCAPP 是一种避免任何单元格修改的方法, 以便它可以在主细胞、细胞培养系统甚至组织样本中应用。然而, 由于这一原因, 它需要将样品交联, 从而降低...

在过去的十年中, 测序技术有了显著的改进, 允许对大量样本中的各种基因组进行研究, 并具有惊人的分辨率。DNA 元素百科全书 (编码) 联合体是国家卫生研究院国家人类基因组研究所牵头的大规模多机构努力, 它提供了关于个人转录因子如何和其他调控蛋白结合在一起, 并与基因组相互作用。最初的努力的特点是特定的 dna 蛋白相互作用, 由染色质免疫沉淀 (芯片) 评估超过100已知的 dna 结合蛋白 1.替代方法, 如 DNase 足迹2和甲醛辅助隔离的管理元素 (自由放任)3也被用来定位特定区域的基因组相互作用的蛋白质, 但明显的局限性,这些实验方法不能识别相互作用的蛋白质。尽管过去几年进行了广泛的努力, 但没有任何技术能有效地使染色质中蛋白质-DNA 相互作用的综合特征, 以及染色质相关蛋白的鉴定和定量化。
为了解决这一需要, 我们开发了一种新的方法, 我们称之为杂交捕获的染色质相关蛋白的蛋白质组学 (HyCCAPP)。最初开发的酵母4,5,6, 该方法分离的交联染色质区域的利益 (与绑定蛋白) 使用序列特定的杂交捕获。在分离蛋白质-DNA 复合物后, 可以用质谱等方法来表征与兴趣序列有关的蛋白质集。因此, HyCCAPP 可以被认为是一种无偏见的方法来揭示新的 DNA-蛋白质相互作用, 在这个意义上, 它不依赖于抗体, 它是完全不可知的蛋白质, 可能被发现。还有其他方法可以发现新的 DNA 相互作用的蛋白质7, 但大多数依赖于类似芯片的方法8,9,10, 质粒插入11,12, 13、14或具有高拷贝数15的区域。相比之下, HyCCAPP 可以应用于多和单拷贝区域, 并且不需要任何先前关于该区域蛋白质的信息。此外, 虽然上面提到的一些方法有很有价值的特点, 特别是避免了 DNA 蛋白交联反应的需要, 但 HyCCAPP 的独特特点是它可以应用于未修饰细胞中的单拷贝区域, 而且没有任何事先了解假定的结合蛋白, 或可用的抗体。
在这一点上, HyCCAPP 主要用于分析酵母中的不同基因组区域4,5,6, 最近被用于分析α卫星 dna 中的蛋白质-DNA 相互作用, 重复区域在人类基因组16中。作为我们正在进行的工作的一部分, 我们已经适应了最初开发的酵母染色质的杂交捕获方法, 适用于人体细胞的分析, 并提出了一个修改的协议, 允许选择性捕获单拷贝目标人类基因组中的区域, 其效率与我们在酵母中的初步研究相似。这种新的优化协议现在允许技术的适应和利用, 通过质谱或其他分析方法来审问人类基因组中的蛋白质-DNA 相互作用。
重要的是要强调, HyCCAPP 方法是为了分析特定的目标区域, 还不适合于全基因组的分析。这项技术在处理那些缺乏相互作用的蛋白质信息的区域时尤其有用, 或者当需要对特定基因组轨迹上的相互作用蛋白质进行更全面的深入分析时。HyCCAPP 的目的是揭示 dna 结合蛋白, 但不能准确描述基因组 DNA 中特定的蛋白质结合点。在目前的实施中, 该方法并没有提供有关单个蛋白质的 DNA 结合序列或图案的信息。因此, 它很好地补充了现有的技术, 如自由放任, 并可能允许识别新的结合蛋白在基因组区域由初步的自由放任分析确定。

<table><tbody><tr><td>PrimerQuest tool</td><td>IDT</td><td></td><td></td></tr><tr><td>OligoAnalyzer 3.1</td><td>IDT</td><td></td><td>Analyze capture oligonucleotids</td></tr><tr><td>PairFold</td><td>UBC</td><td></td><td>Analyze interactions</td></tr><tr><td>RPMI 1640 media</td><td>Thermo Fisher</td><td>11875093</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Thermo Fisher</td><td>25030081</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher</td><td>26140079</td><td></td></tr><tr><td>Countess automated cell counter</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>850 cm&lt;sup&gt;2&lt;/sup&gt; roller bottle&amp;nbsp;</td><td>Greiner Bio-one</td><td>680058</td><td></td></tr><tr><td>Roller system</td><td>Wheaton</td><td>22-288-525</td><td></td></tr><tr><td>37 % formaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Igepal CA-630</td><td>Sigma-Aldrich</td><td>I3021</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P4380</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher</td><td>15630080</td><td></td></tr><tr><td>Branson digital sonifier SFX 150</td><td>Emerson</td><td></td><td></td></tr><tr><td>Qubit 3.0 fluorometer</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Qubit dsDNA BR assay kit</td><td>Thermo Fisher</td><td>Q32850</td><td></td></tr><tr><td>Bioanalyzer 2100</td><td>Agilent</td><td></td><td></td></tr><tr><td>Agilent DNA 1000 kit</td><td>Agilent</td><td>5067-1504</td><td></td></tr><tr><td>MES sodium salt</td><td>Sigma-Aldrich</td><td>M3885</td><td></td></tr><tr><td>NaCl 5M</td><td>Thermo Fisher</td><td>AM9760G</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>DynaMag-2 magnet</td><td>Thermo Fisher</td><td>12321D</td><td></td></tr><tr><td>DynaMag-15 magnet</td><td>Thermo Fisher</td><td>12301D</td><td></td></tr><tr><td>Dynabeads M-280 atreptavidin</td><td>Thermo Fisher</td><td>60210</td><td></td></tr><tr><td>Low binding tubes</td><td>Eppendorf</td><td>22431081</td><td></td></tr><tr><td>Hybridization oven</td><td>SciGene</td><td></td><td></td></tr><tr><td>Tube shaker and rotator</td><td>Thermo Fisher</td><td>415110Q</td><td></td></tr><tr><td>DNase I (RNase-free)</td><td>New England BioLabs</td><td>M0303</td><td></td></tr><tr><td>SSC buffer 20&amp;times; concentrate</td><td>Sigma-Aldrich</td><td>S6639</td><td></td></tr></tbody></table>

adaptation of hybridization capture of chromatin-associated proteins for proteomics to mammalian cells

初步开发了用于蛋白质组学 (HyCCAPP) 技术的染色质相关蛋白的杂交捕获, 以揭示酵母中新的 DNA-蛋白质相互作用。它允许对目标区域进行分析, 而不需要事先了解绑定到目标区域的可能蛋白质。理论上, 这使得 HyCCAPP 可以用来分析任何感兴趣的基因组区域, 并且它提供了足够的灵活性, 可以在不同的细胞系统中工作。这种方法并不意味着研究已知转录因子的结合点, 这项任务更适合于染色质免疫沉淀 (芯片) 和类似芯片的方法。HyCCAPP 的力量在于它探索 DNA 区域的能力, 因为它有有限的或不了解与之相关的蛋白质。它也可以是一种方便的方法, 以避免偏见 (目前在芯片样的方法) 引入蛋白质的染色质浓缩使用抗体。潜在的, HyCCAPP 可以是一个强大的工具, 发现真正的新的 DNA 蛋白相互作用。迄今为止, 该技术主要应用于酵母细胞或哺乳动物细胞中的高复制重复序列。为了成为我们设想的强大工具, HyCCAPP 方法需要进行优化, 以有效地捕获哺乳动物细胞中的单拷贝基因座。在这里, 我们提出了我们对人类细胞系的初始酵母 HyCCAPP 捕获协议的适应, 并表明, 单拷贝染色质区域可以有效地与此修改的协议隔离。

由于需要大量的 HyCCAPP 的染色质来获得成功, 细胞生长到相对较高的融合水平。台盼蓝染色用于确认细胞死亡率适中 (< 10%)。在单拷贝实验中, 杂交捕获之前的染色质含量需要在 femtomolar 范围内, 通常需要至少10个9细胞作为起始材料。在进行全面实验之前, 建议在较小的批次中测试捕获寡核苷酸的性能。在图 1中显示了使用寡核苷酸滴定的杂交?...

Watch this Scientific Journal Video about Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells at JoVE.com

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

这是一种方法, 以确定新的 DNA 相互作用的蛋白质在特定的靶点, 依赖序列特定捕获的交联染色质的后续蛋白质组分析。不需要事先了解潜在的结合蛋白, 也无需进行细胞修饰。该技术最初是为酵母开发的, 现在已经适应了哺乳动物细胞。

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

adaptation-hybridization-capture-chromatin-associated-proteins-for

Research

JoVE Journal

Genetics

6.2K 次观看.  Texas Biomedical Research Institute. 这是一种方法, 以确定新的 DNA 相互作用的蛋白质在特定的靶点, 依赖序列特定捕获的交联染色质的后续蛋白质组分析。不需要事先了解潜在的结合蛋白, 也无需进行细胞修饰。该技术最初是为酵母开发的, 现在已经适应了哺乳动物细胞。

视频: Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

遗传学

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

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 ...

医学

组蛋白组学分析和翻译后修饰表征

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

作者聚焦： 通过 LC-TIMS-TOF MS/MS 和 PASEF 增强组蛋白 PTM 异构体鉴定

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

使用液相色谱法、捕获离子淌度谱法和飞行时间质谱法进行组蛋白修饰筛选

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

化学

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

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

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 ...

生物学

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

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此视频中的章节

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

序贯盐萃取法分析染色质修饰配合物的体染色质结合特性

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

在疾病的染色质蛋白质组的定量分析

使用液相色谱法、捕获离子淌度谱法和飞行时间质谱法进行组蛋白修饰筛选

使用液相色谱法、捕获离子淌度谱法和飞行时间质谱法进行组蛋白修饰筛选

使用液相色谱法、捕获离子淌度谱法和飞行时间质谱法进行组蛋白修饰筛选

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

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术