Name: simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms
Uploaded: 2017-08-10T12:30:00.000+00:00
Duration: 9 min 20 s
Description: Watch this Scientific Journal Video about Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS at JoVE.com

作者承认成本行动 CM1406 （表观遗传化学生物学）。研究报告在这份出版物由皮埃尔 · 米谢尔基金会支持。

1.细胞培养
注: 在标准组培罩执行以下步骤。不育技术熟悉预计。
<ol>
	<li>文化 80%融合在 T75 细胞培养瓶中补充含 10%胎牛血清，青霉素 G 的 MEM HeLa 细胞 (100 U/mL)，和链霉素 （100 毫克/毫升）。 注: 细胞培养、 治疗和裂解步骤在层流流动，与细胞孵育步骤在执行 5%CO2在无菌条件下湿大气中的 37 ° C 下进行。</li>
	<li>洗细胞两次将 5 毫升、 吸出新方案，和添加 1 毫升的细胞分离试剂。5 分钟来分离细胞，37 ℃ 培养。</li>
	<li>添加 9 毫升的 MEM 培养基、 彻底重悬细胞，和用血球计数器单元格进行计数。</li>
	<li>调整到 MEM 6 x 104细胞/毫升细胞悬浮液的密度。</li>
	<li>向控件添加 100 μ L 细胞悬液并测试井的不育、 平底、 组培治疗 96 孔板 （图 1A）。</li>
	<li>将 100 μ L 的 MEM 培养基添加到空白井的 96 孔板 (图 1A)。</li>
	<li>孵育 96 孔板在 37 ° C 为 24 小时。 注意: 24 小时孵化是适当时候，以便在 HeLa 细胞中的内源性 HDAC 活性的试验。不同的细胞类型与未知 HDAC 活性可能需要控制细胞 timecourse 评价。</li>
</ol>
2.细胞治疗试验化合物与 HDAC 基板
<ol>
	<li>吸出的 96 孔板油井介质。</li>
	<li>添加 25 µ L 的 2 x 测试化合物(例如，曲古菌素 A，MS275 和 tubastatin A) 在 MEM 测试井。添加到控制和空白井的 MEM 25 微升。 注: 测试化合物的浓度是进行调整，以提供一系列的至少 5 最终测试浓度 （井每一个浓度）。二甲基亚砜中复合测试最终的含量应不超过 0.5%，应该是相同的每个井，包括空格和控制。最后孵化量每井 50 µ L。在从 500 到 1.95 5 浓度测试曲毫微米 （1:4 稀释）。MS275 和 tubastatin A 测试 5 浓度从 8000 至 6.17 毫微米 （1:6 稀释）。对于每个测试复合所需的最后测试浓度应测定每个用户通过创建一个剂量-反应曲线。</li>
	<li>添加到控制和测试井 25 微升的基材混合物 (仲裁法 》、 42 µ M 的 MOCPAC，和 BATCP 在 MEM 42 μ M 42 µ M)。添加空白井的 MEM 25 微升。 注: 最终每个底物浓度的是 MOCPAC 和 BATCP 分别区分 HDAC1 和 HDAC6 的活动，(图 1B)。 21 µ M。当没有活动观察那些基板时，仲裁法 》 的使用可能潜在活动反对另一个 HDAC 异构体的标识了。</li>
	<li>孵育 96 孔板在 37 ° C 的湿化液的 5 %8 h CO2孵化器。 注: 此孵育时间适合 HeLa 细胞和允许内源性的 HDAC 与合成基质进行交互。不同的细胞株可能需要在孵化时间和/或底物浓度的调整。</li>
</ol>
3.细胞裂解和 UHPLC-质谱-质谱样品制备
注意: 样品制备步骤使用有机化学品，是高度易燃和有毒通过摄入或吸入。穿适当的个人防护，如手套、 安全眼镜。
<ol>
	<li>添加 10 微升 6 x RIPA 缓冲区 （从 10 x RIPA 缓冲液稀释） 辅以每口井的 96 孔板的蛋白酶抑制剂。</li>
	<li>通过添加每口井的冷乙腈 160 µ L 停止反应和混合吹打向上和向下。 注意: 在-20 ° C 在这一步之前保持乙腈。</li>
	<li>将盘子放在-80 ° C 冰箱 10 分钟。</li>
	<li>从冰箱里删除板和 220 µ L 的每口井的内容转移到非无菌的锥底 （V 形底） 96 孔板。离心板 5000 x g 和 10 分钟 4 ° C。 注: 此步骤旨在消除蛋白质和盐。如有必要，解决方案可以转移到个人的锥形离心管前离心或过滤通过一套 0.65 微米聚偏氟乙烯滤板 96 孔板。成功清除的沉淀是需要在 UHPLC 分析之前。</li>
	<li>上清液 （或过滤） 200 微升转入 96 孔板与 UHPLC 系统兼容和密封用可剥性、 热封箔使用板封口机。 注意: 避免吹打颗粒时去除上清液。如果无法立即执行 UHPLC 分析，板可以直到分析存储在 4 ° C，最长为 24 小时。</li>
</ol>
4.UHPLC/质谱-质谱分析
<ol>
	<li>来填补它与流动相系统 （95%A:5 %b) 准备 UHPLC 系统: 答: H2O，高效液相色谱级含 0.1%甲酸 （准备 1 L） B: 乙腈、 高效液相色谱法等级、 含 0.1%甲酸 （准备 1 升）。 注意: 移动相含有有机化学品，是高度易燃和有毒通过摄入或吸入。穿适当的个人防护，如手套、 安全眼镜。</li>
	<li>放入示例管理器包含一个板持有人 96 孔板。运行根据表 1中提供的 UHPLC-质谱-质谱条件示例。 注意: 分析 96 孔板每一列应该有一个控制井和一个空白井 （板方案的图 1A所示）。控件是活性的代表 100%测试的细胞系中，酶而空白有检查是否 MS 源活性的提供一致的背景。在一个给定的空白中检测到的不寻常 MS 峰应调查以防止 MS 灵敏度变化。</li>
</ol>
5.数据分析
<ol>
	<li>与适当的 UHPLC 集成软件集成山顶区的每个底物和其脱乙酰基的产物。 注意: 使用自动集成参数平滑法 （均值、 窗口大小 3 和数目光滑 2，例如）。使用表 1中规定的色谱条件，洗脱顺序是 dMAL （5.8 分钟）、 dBATCP （6.0 分）、 dMOCPAC （6.2 分）、 仲裁法 》 （7.6 分钟）、 MOCPAC （8.9 分） 和 BATCP （9.8 分钟），如图 1所示。</li>
	<li>对于每种基材，计算峰面积比值 （脱乙酰/乙酰化） 在每个色谱 （测试和控制样品）。</li>
	<li>计算每个测试样本的 HDAC 抑制百分比: 
	<img alt="Equation" src="/files/ftp_upload/55878/55878eq1.jpg"> 注: HDAC1 抑制使用计算峰面积比值的 dMOCPAC/MOCPAC;HDAC6 抑制被计算 dBATCP/BATCP。可以用比 dMAL / 仲裁法 》，来表达一般 HDAC 抑制。</li>
</ol>

<ol>
	<li>Arrowsmith, C. H., Bountra, C., Fish, P. V., Lee, K., Schapira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+protein+families:+a+new+frontier+for+drug+discovery.">Epigenetic protein families: a new frontier for drug discovery.</a> Nat Rev Drug Discov. 11 (5), 384-400 (2012).</li><li>Henikoff, S., Greally, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics,+cellular+memory+and+gene+regulation.">Epigenetics, cellular memory and gene regulation.</a> Curr Biol. 26 (14), R644-R648 (2016).</li><li>Kim, 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=HDAC6+inhibitor+blocks+amyloid+beta-induced+impairment+of+mitochondrial+transport+in+hippocampal+neurons.">HDAC6 inhibitor blocks amyloid beta-induced impairment of mitochondrial transport in hippocampal neurons.</a> PLoS One. 7 (8), (2012).</li><li>Kawaguchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+deacetylase+HDAC6+regulates+aggresome+formation+and+cell+viability+in+response+to+misfolded+protein+stress.">The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress.</a> Cell. 115 (6), 727-738 (2003).</li><li>Zhao, Y., 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=Acetylation+of+p53+at+lysine+373/382+by+the+histone+deacetylase+inhibitor+depsipeptide+induces+expression+of+p21(Waf1/Cip1).">Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1).</a> Mol Cell Biol. 26 (7), 2782-2790 (2006).</li><li>Berger, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+modifications+in+transcriptional+regulation.">Histone modifications in transcriptional regulation.</a> Curr Opin Genet Dev. 12 (2), 142-148 (2002).</li><li>Sim&#245;es-Pires, 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=HDAC6+as+a+target+for+neurodegenerative+diseases:+what+makes+it+different+from+the+other+HDACs?.">HDAC6 as a target for neurodegenerative diseases: what makes it different from the other HDACs?.</a> Mol Neurodegener. 8 (7), 1-16 (2013).</li><li>Mottamal, M., Zheng, S., Huang, T. L., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+in+clinical+studies+as+templates+for+new+anticancer+agents.">Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents.</a> Molecules. 20 (3), 3898-3941 (2015).</li><li>Manal, M., Chandrasekar, M. J., Gomathi Priya, J., Nanjan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+histone+deacetylase+as+antitumor+agents:+A+critical+review.">Inhibitors of histone deacetylase as antitumor agents: A critical review.</a> Bioorg Chem. 67, 18-42 (2016).</li><li>Mack, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+selectivity+and+beyond.">To selectivity and beyond.</a> Nat Biotech. 28 (12), 1259-1266 (2010).</li><li>. Study of ACY-1215 alone and in combination with bortezomib and dexamethasone in multiple myeloma (ACY-1215) Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01323751">https://clinicaltrials.gov/ct2/show/NCT01323751</a> (1211)</li><li>. Study of ACY-1215 in combination with lenalidomide, and dexamethasone in multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01583283">https://clinicaltrials.gov/ct2/show/NCT01583283</a> (2012)</li><li>. 13ACY-1215 (ricolinostat) in combination with pomalidomide and low-dose dex in relapsed-and-refractory multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01997840">https://clinicaltrials.gov/ct2/show/NCT01997840</a> (2013)</li><li>. Study of ACY-241 alone and in combination with pomalidomide and dexamethasone in multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT02400242">https://clinicaltrials.gov/ct2/show/NCT02400242</a> (2015)</li><li>Yee, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ricolinostat+plus+lenalidomide,+and+dexamethasone+in+relapsed+or+refractory+multiple+myeloma:+a+multicentre+phase+1b+trial.">Ricolinostat plus lenalidomide, and dexamethasone in relapsed or refractory multiple myeloma: a multicentre phase 1b trial.</a> Lancet Oncol. 17 (11), 1569-1578 (2016).</li><li>Jung, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogenous+non-isotopic+assays+for+histone+deacetylase+activity.">Homogenous non-isotopic assays for histone deacetylase activity.</a> Expert Opin Ther Pat. 13 (6), 935 (2003).</li><li>Zwick, V., Sim&#245;es-Pires, C., Cuendet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+multi-substrate+assay+coupled+to+UHPLC-ESI-MS/MS+for+a+quick+identification+of+class-specific+HDAC+inhibitors.">Cell-based multi-substrate assay coupled to UHPLC-ESI-MS/MS for a quick identification of class-specific HDAC inhibitors.</a> J Enzyme Inhibi Med Chem. 31 (1), 209-214 (2016).</li><li>Khan, N., 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=Determination+of+the+class+and+isoform+selectivity+of+small-molecule+histone+deacetylase+inhibitors.">Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors.</a> Biochem J. 409 (2), 581-589 (2008).</li><li>Glaser, K. 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=Differential+protein+acetylation+induced+by+novel+histone+deacetylase+inhibitors.">Differential protein acetylation induced by novel histone deacetylase inhibitors.</a> Biochem Biophys Res Commun. 325 (3), 683-690 (2004).</li><li>Butler, K. V., 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=Rational+design+and+simple+chemistry+yield+a+superior,+neuroprotective+HDAC6+inhibitor,+tubastatin+A.">Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A.</a> J Am Chem Soc. 132 (31), 10842-10846 (2010).</li><li>Jung, M., Yong, K. -. J., Velena, A., Lee, S., Sippl, W., Jung, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Assays+for+HDAC+Inhibitor+Hit+Validation.">Cell-Based Assays for HDAC Inhibitor Hit Validation.</a> Epigenetic Targets in Drug Discovery. , (2010).</li><li>Milli, 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=Proteomic+analysis+of+cellular+response+to+novel+proapoptotic+agents+related+to+atypical+retinoids+in+human+IGROV-1+ovarian+carcinoma+cells.">Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells.</a> J Proteome Res. 10 (3), 1191-1207 (2011).</li><li>Zwick, V., 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=Synthesis+of+a+selective+HDAC6+inhibitor+active+in+neuroblasts.">Synthesis of a selective HDAC6 inhibitor active in neuroblasts.</a> Bioorg Med Chem Lett. 26 (20), 4955-4959 (2016).</li><li>Ciossek, T., Julius, H., Wieland, H., Maier, T., Beckers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homogeneous+cellular+histone+deacetylase+assay+suitable+for+compound+profiling+and+robotic+screening.">A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening.</a> Anal Biochem. 372 (1), 72-81 (2008).</li><li>Heltweg, B., Dequiedt, F., Marshall, B. L., Brauch, C., Yoshida, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype+selective+substrates+for+histone+deacetylases.">Subtype selective substrates for histone deacetylases.</a> J Med Chem. 47 (21), 5235-5243 (2004).</li><li>Copeland, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evaluation of enzyme inhibitors in drug discovery. , (2005).</li></ol>

作者没有透露。

HDAC 抑制是一个热门话题，药物发现，当前的重点放在为癌症治疗21HDAC6 选择性抑制剂。HDAC 选择性通常是由一系列的高吞吐量、 无细胞化验，打算对个别 HDAC 异构体21抑制效价评定的。然而，一种抑制剂的选择性必须另外确认在活细胞中通过评估内源性蛋白质底物，如组蛋白的乙酰化状态 （为 I 类乙酰） 和微管蛋白 （为 HDAC6)。因此，细胞化验涉及抗体识别 ?...

乙酰属于一个家庭能够脱乙酰度内的染色质结构的组蛋白的酶。他们还在胞浆内有其他蛋白质底物和位于中各单元格舱室。共 18 HDAC 异构体迄今为止识别出已涉及到几种细胞机制，包括转录因子基因的表达，以及细胞信号转导的调控和运输1，，23，4，5，，67。催化 HDAC 抑制剂已成为潜在的治疗药物，用于治疗癌症。大多数的 HDAC 抑制剂，目前被 FDA 批准为 T 细胞淋巴瘤和多发性骨髓瘤的治疗，非特定 HDAC 抑制剂，如 vorinostat (SAHA)、 belinostat 和 panobinostat8，9。然而，一系列的副作用已与泛-抑制剂和亚型特异性小分子搜索是一个热门话题，在药物化学、 新药开发。因此，类我 （HDAC1 3 和 HDAC8） 选择性抑制剂 romidepsin 是已经核准的药物10，而 HDAC6 特异性抑制剂，目前正在进行的临床实验，在多发性骨髓瘤11，，1213，，1415增加治疗潜力。
筛查检测来表征 HDAC 抑制剂都基于 HDAC 基板与酶源 （单一异构体、 核提取物或细胞裂解液） 的孵化。承印物上通常是含耦合到裂解荧光 （例如，香豆素），乙酰基赖氨酸残基的小分子多肽序列，如 N-(4-methyl-7-aminocoumarinyl)-Nα-(t-butoxycarbonyl)-Nω-acetyllysineamide (MAL)16。为了区分异构体的具体活动，涉及每个异构体的分离无细胞化验是必要和可能不会反映在活细胞中的真正异构体活动。异构体特定底物市面上，如苄基 (S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate (MOCPAC，HDAC1 具体基板) 和 (S)-[5-acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamic 酸叔丁酯 (BATCP，HDAC6 具体基板) (图 1B)。然而，仲裁法 》 的多基质混合物中，MOCPAC 和 BATCP 给活细胞将不允许个别的脱乙酰度产品检测荧光法测定，既然他们承担相同的裂解荧光。
这里介绍的方法可以检测和相对定量方法的每个衬底和 HeLa 细胞使用紧接着 UHPLC-质谱-质谱分析17多底物测定其脱乙酰基的产品。HDAC 检测被进行对 HeLa 细胞能够直接识别的 HDAC 抑制活性和特异性的测试化合物对内源性乙酰。那里是专注于 HDAC1 和 HDAC6，同时评估。为了实现在单一孵化测定这些酶法测量，非异性和特异性 HDAC 基质混合物被添加到在 96 孔板上镀的处理和未经处理 HeLa 细胞。后孵化的一步，细胞的裂解释放基质和他们各自的反应产物，这分离和检测 UHPLC 气相色谱-质谱法 (图 1)。仲裁法 》，MOCPAC 和 BATCP 的底物的脱乙酰度的产品分别是脱乙酰度的玛 (dMAL)、 脱乙酰度的 MOCPAC (dMOCPAC) 和脱乙酰度的 BATCP (dBATCP)。剂量-反应曲线可以用活性的化合物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55878/55878fig1.jpg"> 
图 1: 基于单元格的 HDAC 法通过多个基板 UHPLC 气相色谱-质谱分析鉴定特定于 HDAC1 和 HDAC6 抑制剂的一般计划。(A) 计划的典型 96 孔板化合物治疗 （测试） 和 HeLa 细胞 （对照），以及无细胞的空白。(B) 补充说: 作为一种混合物 (每个 21 µ M)，由内源性乙酰脱乙酰基底的化学结构。(C) 典型 UHPLC-质谱联用色谱显示添加衬底 （仲裁法 》，MOCPAC 和 BATCP） 和脱乙酰度的产品的峰值 (dMAL，dMOCPAC，和 dBACTP，分别)。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55878/55878fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

<table><tbody><tr><td>BATCP</td><td>Sigma-Aldrich</td><td>B4061</td><td></td></tr><tr><td>MAL</td><td>Sigma-Aldrich</td><td>SCP0168</td><td>Synonym: BOC-Ac-Lys-AMC</td></tr><tr><td>MOCPAC</td><td>Sigma-Aldrich</td><td>M2195</td><td></td></tr><tr><td>MS275</td><td>Sigma-Aldrich</td><td>EPS002</td><td></td></tr><tr><td>Trichostatin A</td><td>Sigma-Aldrich</td><td>T8552</td><td></td></tr><tr><td>Tubastatin A</td><td>Sigma-Aldrich</td><td>SML0044</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher Scientific</td><td>10660131</td><td></td></tr><tr><td>Formic acid, LC/MS grade</td><td>Fisher Scientific</td><td>10596814</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, HPLC grade</td><td></td><td></td><td>distilled H&lt;sub&gt;2&lt;/sub&gt;O filtered through a Milli-Q purification system</td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>ATCC CRM-CCL-2</td><td></td></tr><tr><td>Cell dissociation reagent TrypLE Express</td><td>ThermoFisher Scientific</td><td>12604013</td><td></td></tr><tr><td>DMSO, cell culture grade</td><td>Applichem</td><td>146463</td><td></td></tr><tr><td>DPBS</td><td>ThermoFisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Biowest</td><td>S1810</td><td></td></tr><tr><td>MEM</td><td>ThermoFisher Scientific</td><td>22561021</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Bioconcept</td><td>4-01F00-H</td><td></td></tr><tr><td>10x RIPA buffer</td><td>Abcam</td><td>ab156034</td><td></td></tr><tr><td>SigmaFast protease inhibitor tablets</td><td>Sigma-Aldrich</td><td>S8820</td><td></td></tr><tr><td>96-well plates, sterile, flat-bottom, tissue culture treated Corning</td><td>VWR</td><td>29442-058</td><td></td></tr><tr><td>96-well plates, non-sterile, V-bottom Corning</td><td>VWR</td><td>29442-404</td><td>used in the centrifugation step</td></tr><tr><td>96-well plate, conical bottom, Nunc</td><td>ThermoFisher Scientific</td><td>249944</td><td>compatible with the Acquity UHPLC system</td></tr><tr><td>T75 cell culture flasks Corning</td><td>Sigma-Aldrich</td><td>CLS430641</td><td></td></tr><tr><td>Peelable heat sealing foil</td><td>Waters</td><td>186002789</td><td></td></tr><tr><td>Acquity UPLC system</td><td>Waters</td><td></td><td></td></tr><tr><td>Eppendorf Centrifuge 5810 R</td><td>Fisher Scientific</td><td>05-413-323</td><td></td></tr><tr><td>Integration software: MassLynx V4.1</td><td>Waters</td><td></td><td>Catalog number not available</td></tr><tr><td>Combi thermo-sealer SP-0669/240</td><td>Waters</td><td></td><td>Catalog number not available</td></tr><tr><td>Quattro micro API Tandem Quadrupole System</td><td>Waters</td><td></td><td>Catalog number not available</td></tr></tbody></table>

simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms

寻找新的组蛋白去乙酰化酶 (HDAC) 抑制剂是提高药物发现感兴趣。异构体选择性以来，在聚光灯下的 romidepsin，一类批准我 HDAC 抑制剂用于癌症治疗和 HDAC6 特异性抑制剂对多发性骨髓瘤的临床研究。本方法用于确定测试化合物 HDAC1 和 HDAC6 细胞的抑制活性。使用超高性能液相色谱 — — 质谱 (UHPLC-MS) 分析方法对特定的底物与处理和未经处理的 HeLa 细胞培养，测定了异构体活性。该方法的优点是反映内的细胞环境，与无细胞的生化研究孤立异构体的内源性 HDAC 活动。此外，因为它基于定量化合成基质，方法不需要内源性乙酰化蛋白抗体识别。它很容易适用于几种细胞系和一个自动化的过程。该方法已经证明有用找到 HDAC6 选择性化合物在神经母细胞。代表性的结果如下所示的标准的 HDAC 抑制剂曲 （非特定），MS275 （特定的 HDAC1） 和 tubastatin （HDAC6-特定） 使用 HeLa 细胞。

能够识别中的应用方法非选择性和选择性抑制剂为 HDAC1 （第一类） 和 HDAC6 （类 IIb），HeLa 细胞治疗与已知的标准化合物: 曲古菌素 A （非选择性 HDAC1 和 HDAC6 之间）18、19MS275 （HDAC1 选择性抑制剂） 和 tubastatin （HDAC6 选择性抑制剂）20。使用本方法 (图 1)，IC50值可以直接确定在活细胞中 HD...

Watch this Scientific Journal Video about Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS at JoVE.com

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

本方法用于确定亚型特异性抑制剂组蛋白去乙酰化酶 (HDAC) 在 HeLa 细胞中由多个基板 UHPLC 气相色谱-质谱分析。这是一种抗体免费的方法，开发以反映生活的 HDAC1 和 HDAC6 活性与单一异构体无细胞检测的细胞环境。

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

simultaneous-measurement-hdac1-hdac6-activity-hela-cells-using-uhplc

Universidad San Sebastian

Research

JoVE Journal

Chemistry

8.5K 次观看.  Universités des Geneva–Lausanne (EPGL). 本方法用于确定亚型特异性抑制剂组蛋白去乙酰化酶 (HDAC) 在 HeLa 细胞中由多个基板 UHPLC 气相色谱-质谱分析。这是一种抗体免费的方法，开发以反映生活的 HDAC1 和 HDAC6 活性与单一异构体无细胞检测的细胞环境。

视频: Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

科研

生物化学

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

验证组蛋白乙酰转移酶抑制剂的检测

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

癌症研究

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

开发基于点击的检测方法，用于筛选 HAT1 酶活性

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

作者聚焦:开发用于 HAT1 抑制剂筛选的乙酰点击检测

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

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 ...

生物学

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

h3 和 h4 组蛋白的纯化及细胞和脑组织中乙酰化组蛋白标记的定量

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

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此视频中的章节

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

验证组蛋白乙酰转移酶抑制剂的检测

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

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

哺乳动物链接组蛋白亚型表达分析

h3 和 h4 组蛋白的纯化及细胞和脑组织中乙酰化组蛋白标记的定量