Name: isolation of histone from sorghum leaf tissue for top down mass spectrometry profiling of potential epigenetic markers
Uploaded: 2021-03-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers at JoVE.com

我们感谢罗纳德·摩尔和托马斯·菲尔莫尔帮助进行质谱学实验，感谢马修·梦露的数据沉积。这项研究由美国能源部（DOE）生物和环境研究资助，通过高梁（EPICON）的表观遗传控制抗旱项目，获得美国农业部（USDA）颁发的编号为DE-SC0014081的奖项：CRIS 2030-21430-008-00D），并通过联合生物能源研究所（JBEI），由能源部（DE-AC02-05CH11231）赞助的设施劳伦斯伯克利国家实验室和能源部。这项研究是利用环境分子科学实验室（EMSL）（网格.436923.9）进行的，该实验室是由生物和环境研究办公室赞助的能源部科学用户设施办公室。

1. 准备高梁叶材料
注：高梁植物生长在加州帕利尔的土壤中。
<ol><li>将植物中的高梁叶收集到50mL离心管中，并立即将管子冷冻在液氮中。通过从主耕种机中撕下第三和第四完全脱落的叶子来收集叶组织。 注：有关现场状况、样本生长和收集的更多详细信息，请在已发布的报告18中找到。</li>
	<li>用液氮磨叶，并立即转移到离心管。</li>
	<li>将地叶存放在-...

<ol>
	<li>Farooq, M., Wahid, A., Kobayashi, N., Fujita, D., Basra, S. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+drought+stress:+Effects,+mechanisms+and+management.">Plant drought stress: Effects, mechanisms and management.</a> Agronomy for Sustainable Development. , 153-188 (2009).</li><li>Dai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drought+under+global+warming:+a+review.">Drought under global warming: a review.</a> Wiley Interdisciplinary Reviews: Climate Change. 2 (1), 45-65 (2011).</li><li>Rooney, W. L., Blumenthal, J., Bean, B., Mullet, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+sorghum+as+a+dedicated+bioenergy+feedstock.">Designing sorghum as a dedicated bioenergy feedstock.</a> Biofuels, Bioproducts and Biorefining. 1 (2), 147-157 (2007).</li><li>Mullet, J. E., Klein, R. R., Klein, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorghum+bicolor+-+an+important+species+for+comparative+grass+genomics+and+a+source+of+beneficial+genes+for+agriculture.">Sorghum bicolor - an important species for comparative grass genomics and a source of beneficial genes for agriculture.</a> Current Opinion in Plant Biology. 5 (2), 118-121 (2002).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drought+delays+development+of+the+sorghum+root+microbiome+and+enriches+for+monoderm+bacteria.">Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (18), 4284-4293 (2018).</li><li>Gao, 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=Strong+succession+in+arbuscular+mycorrhizal+fungal+communities.">Strong succession in arbuscular mycorrhizal fungal communities.</a> ISME Journal. 13 (1), 214-226 (2019).</li><li>Gao, 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=Fungal+community+assembly+in+drought-stressed+sorghum+shows+stochasticity,+selection,+and+universal+ecological+dynamics.">Fungal community assembly in drought-stressed sorghum shows stochasticity, selection, and universal ecological dynamics.</a> Nature Communications. 11 (1), (2020).</li><li>Bannister, A. J., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chromatin+by+histone+modifications.">Regulation of chromatin by histone modifications.</a> Cell Research. 21 (3), 381-395 (2011).</li><li>Yuan, L., Liu, X., Luo, M., Yang, S., Wu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+histone+modifications+in+plant+abiotic+stress+responses.">Involvement of histone modifications in plant abiotic stress responses.</a> Journal of Integrative Plant Biology. 55 (10), 892-901 (2013).</li><li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nature Reviews. Genetics. 10 (10), 669-680 (2009).</li><li>Huang, H., Lin, S., Garcia, B. A., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+analysis+of+histone+modifications.">Quantitative proteomic analysis of histone modifications.</a> Chemical Reviews. 115 (6), 2376-2418 (2015).</li><li>Moradian, A., Kalli, A., Sweredoski, M. J., Hess, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+top-down,+middle-down,+and+bottom-up+mass+spectrometry+approaches+for+characterization+of+histone+variants+and+their+post-translational+modifications.">The top-down, middle-down, and bottom-up mass spectrometry approaches for characterization of histone variants and their post-translational modifications.</a> Proteomics. 14 (4-5), 489-497 (2014).</li><li>Sidoli, S., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+individual+histone+posttranslational+modifications+and+their+combinatorial+patterns+by+mass+spectrometry-based+proteomics+strategies.">Characterization of individual histone posttranslational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies.</a> Methods in Molecular Biology. 1528, 121-148 (2017).</li><li>Maile, T. 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=Mass+spectrometric+quantification+of+histone+post-translational+modifications+by+a+hybrid+chemical+labeling+method.">Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.</a> Molecular &amp; Cellular Proteomics. 14 (4), 1148-1158 (2015).</li><li>Dang, 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=The+first+pilot+project+of+the+consortium+for+top-down+proteomics:+a+status+report.">The first pilot project of the consortium for top-down proteomics: a status report.</a> Proteomics. 14 (10), 1130-1140 (2014).</li><li>Schaffer, L. 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=Identification+and+quantification+of+proteoforms+by+mass+spectrometry.">Identification and quantification of proteoforms by mass spectrometry.</a> Proteomics. 19 (10), 1800361 (2019).</li><li>Cupp-Sutton, K. A., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+quantitative+top-down+proteomics.">High-throughput quantitative top-down proteomics.</a> Molecular Omics. , (2020).</li><li>Varoquaux, 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=Transcriptomic+analysis+of+field-droughted+sorghum+from+seedling+to+maturity+reveals+biotic+and+metabolic+responses.">Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (52), 27124 (2019).</li><li>Gordon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+vanadate+as+protein-phosphotyrosine+phosphatase+inhibitor.">Use of vanadate as protein-phosphotyrosine phosphatase inhibitor.</a> Methods in Enzymology. 201, 477-482 (1991).</li><li>Zhou, 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=Profiling+changes+in+histone+post-translational+modifications+by+top-down+mass+spectrometry.">Profiling changes in histone post-translational modifications by top-down mass spectrometry.</a> Methods in Molecular Biology. 1507, 153-168 (2017).</li><li>Chambers, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cross-platform+toolkit+for+mass+spectrometry+and+proteomics.">A cross-platform toolkit for mass spectrometry and proteomics.</a> Nature Biotechnology. 30 (10), 918-920 (2012).</li><li>Kou, Q., Xun, L., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopPIC:+a+software+tool+for+top-down+mass+spectrometry-based+proteoform+identification+and+characterization.">TopPIC: a software tool for top-down mass spectrometry-based proteoform identification and characterization.</a> Bioinformatics (Ocford, England). 32 (22), (2016).</li><li>Park, 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=Informed-Proteomics:+open-source+software+package+for+top-down+proteomics.">Informed-Proteomics: open-source software package for top-down proteomics.</a> Nature Methods. 14 (9), 909-914 (2017).</li><li>LeDuc, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C-Score:+a+bayesian+framework+to+sharply+improve+proteoform+scoring+in+high-throughput+top+down+proteomics.">The C-Score: a bayesian framework to sharply improve proteoform scoring in high-throughput top down proteomics.</a> Journal of Proteome Research. 13 (7), 3231-3240 (2014).</li><li>Fornelli, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+top-down+analysis+of+the+human+proteome+using+a+benchtop+quadrupole-orbitrap+mass+spectrometer.">Advancing top-down analysis of the human proteome using a benchtop quadrupole-orbitrap mass spectrometer.</a> Journal of Proteome Research. 16 (2), 609-618 (2017).</li><li>Sun, R. 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=pTop+1.0:+A+high-accuracy+and+high-efficiency+search+engine+for+intact+protein+identification.">pTop 1.0: A high-accuracy and high-efficiency search engine for intact protein identification.</a> Analytical Chemistry. 88 (6), 3082-3090 (2016).</li><li>Xiao, K., Yu, F., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+protein+identification+using+isotopic+envelope+fingerprinting.">Top-down protein identification using isotopic envelope fingerprinting.</a> Journal of Proteomics. 152, 41-47 (2017).</li><li>Cai, W., 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=MASH+Suite+Pro:+A+comprehensive+software+tool+for+top-down+proteomics.">MASH Suite Pro: A comprehensive software tool for top-down proteomics.</a> Molecular &amp; Cellular Proteomics: MCP. 15 (2), 703-714 (2016).</li><li>Zhou, 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=Top-down+mass+spectrometry+of+histone+modifications+in+sorghum+reveals+potential+epigenetic+markers+for+drought+acclimation.">Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.</a> Methods. , (2019).</li><li>Garcia, B. A., Pesavento, J. J., Mizzen, C. A., Kelleher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+combinatorial+modification+of+histone+H3+in+human+cells.">Pervasive combinatorial modification of histone H3 in human cells.</a> Nature Methods. 4 (6), 487-489 (2007).</li><li>Zheng, 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=Unabridged+analysis+of+human+histone+H3+by+differential+top-down+mass+spectrometry+reveals+hypermethylated+proteoforms+from+MMSET/NSD2+overexpression.">Unabridged analysis of human histone H3 by differential top-down mass spectrometry reveals hypermethylated proteoforms from MMSET/NSD2 overexpression.</a> Molecular &amp; Cellular Proteomics: MCP. 15 (3), 776-790 (2016).</li><li>Garcia, B. 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=Chemical+derivatization+of+histones+for+facilitated+analysis+by+mass+spectrometry.">Chemical derivatization of histones for facilitated analysis by mass spectrometry.</a> Nature Protocols. 2 (4), 933-938 (2007).</li><li>Holt, M. V., Wang, T., Young, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-pot+quantitative+top-+and+middle-down+analysis+of+GluC-digested+histone+H4.">One-pot quantitative top- and middle-down analysis of GluC-digested histone H4.</a> Journal of the American Society for Mass Spectrometry. 30 (12), 2514-2525 (2019).</li><li>Tian, Z., 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=Enhanced+top-down+characterization+of+histone+post-translational+modifications.">Enhanced top-down characterization of histone post-translational modifications.</a> Genome Biology. 13 (10), (2012).</li><li>Wang, Z., Ma, H., Smith, K., Wu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+separation+using+high-pH+and+low-pH+reversed+phase+liquid+chromatography+for+top-down+proteomics.">Two-dimensional separation using high-pH and low-pH reversed phase liquid chromatography for top-down proteomics.</a> International Journal of Mass Spectrometry. 427, 43-51 (2018).</li><li>Gargano, A. F. G., 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=Increasing+the+separation+capacity+of+intact+histone+proteoforms+chromatography+coupling+online+weak+cation+exchange-HILIC+to+reversed+phase+LC+UVPD-HRMS.">Increasing the separation capacity of intact histone proteoforms chromatography coupling online weak cation exchange-HILIC to reversed phase LC UVPD-HRMS.</a> Journal of Proteome Research. 17 (11), 3791-3800 (2018).</li></ol>

所提交的协议描述了如何从高梁叶（或更普遍的植物叶）样本中提取组织素。平均石材产量预计为每4-5克高梁叶材料2-20微克。这些材料足够纯净，用于LC-MS（主要是带~20%核糖体蛋白污染的组织石）的下游结石分析。由于样本变化或整个协议中可能处理不当/失败，可能会获得较低的产量。在核裂解步骤之前保持核的完整性至关重要：因此，在添加 NLB 之前，应避免攻击性涡流和管道。此外，从颗粒...

干旱的严重程度和频率的增加预计将影响谷物作物的产量1，2。高梁是一种谷物食品和能源作物，以其抗水限水的特殊能力而闻名于世。我们正在对干旱压力、植物发育和高梁（高梁双色（L.）莫恩奇植物的表观遗传学之间的相互作用进行机械理解。我们先前的工作已经证明植物和里佐圈微生物群在干旱适应和分子水平5，6，7的反应之间有很强的联系。这项研究将为利用表观遗传学工程使作物适应未来的气候情景铺平道路。作为理解表观遗传学努力的一部分，我们的目标是研究影响植物生物体内基因表达的蛋白质标记。
组蛋白属于真核生物中高度保存的蛋白质家族，将DNA包装成核糖体作为染色质的基本单位。组蛋白的转化后修改 （PTM） 受到动态调节，以控制色素结构并影响基因表达。像其他表观遗传因素，包括DNA甲基化，石质PTM在许多生物过程8，9起着重要的作用。基于抗体的检测，如西方污点，已被广泛用于识别和量化石斑点。此外，通过色素免疫沉淀——测序（ChIP-seq）10，可以有效地探究组织PTM和DNA的相互作用。在 ChIP-seq 中，具有特定靶向层蛋白 PTM 的色素通过针对该特定 PTM 的抗体而富含。然后，DNA片段可以从富集的染色质中释放并测序。与靶向层石PTM相互作用的基因区域被揭示出来。然而，所有这些实验严重依赖高质量的抗体。对于一些类结石变异/同源或PTM组合，开发强健的抗体可能极具挑战性（尤其是对于多个PTM）。此外，只有当已知目标的石块PTM时，才能开发抗体。11因此，需要采用非目标的全球平石 PTM 分析的替代方法。
质谱学 （MS） 是一种补充方法，用于描述平石 PTM 的特征，包括未知的 PTM，其中抗体不可用11，12。在液相色谱 （LC） 分离和 MS 检测之前，成熟的"自下而上"MS 工作流使用蛋白酶将蛋白质消化成小肽。由于类固酮有大量基本残留物（赖氨酸和精氨酸），标准自下而上的工作流程中的丁普辛消化（赖氨酸和精氨酸特有的蛋白酶）将蛋白质切成非常短的肽。短肽在技术上很难通过标准LC-MS进行分析，并且不保存有关多个PTM的连接性和口感学的信息。使用其他酶或化学标签来阻止赖氨酸产生更长的肽，更适合组别石PTM13，14的特征。
或者，消化步骤可以完全省略。在这种"自上而下"的方法中，完整的蛋白质离子在在线LC分离后通过电喷电离（ESI）引入MS，产生完整的石蛋白形成体的离子。此外，在质谱仪中可以分离和分割感兴趣的离子（即蛋白状体），以产生用于识别和PTM定位的序列离子。因此，自上而下的MS具有保存蛋白突起级信息和捕获多个PTM和终端截断的连接性在同一蛋白回形15，16的优势。自上而下的实验也可以提供定量信息，并提供完整的蛋白质水平17的生物标志物的见解。在这里，我们描述了一个从高梁叶中提取石块并通过自上而下LC-MS分析完整发音的协议。
图 1 和图 2中显示的示例数据来自种植后第 2 周收集的高梁叶。 虽然预期产量会有所变化，但此协议通常对特定的样本条件不可知。同样的协议已经成功地用于高梁植物叶组织收集从种植后2，3，5，8，9和10周。

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Chemical</td>
			<td>A955-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>43815-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 500mM Solution, pH 8.0</td>
			<td>EMD Millipore Corp</td>
			<td>324504-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Thermo Scientific</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine Hydrochloride</td>
			<td>Sigma</td>
			<td>G3272-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic</td>
			<td>Sigma</td>
			<td>P3786-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail, cOmplete tablets</td>
			<td>Roche</td>
			<td>5892791001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium butyrate</td>
			<td>Sigma</td>
			<td>303410-5G</td>
			<td>Used for histone deacetylase inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S7020-100G</td>
			<td>Used for phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>450243-10G</td>
			<td>Used for phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td>BP153-500 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Weak cation exchange resin, mesh 100-200 analytical (BioRex70)</td>
			<td>Bio-Rad</td>
			<td>142-5842</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography column (Bio-Spin)</td>
			<td>BIO-RAD</td>
			<td>732-6008</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh 100 filter cloth</td>
			<td>Millipore Sigma</td>
			<td>NY1H09000</td>
			<td>This is part of the Sigma kit (catalog # CELLYTPN1) for plant nuclei extraction. Similar filters with the same mesh size can be used.</td>
		</tr>
		<tr>
			<td>Micropipette tips (P20, P200, P1000)</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, 50mL/15mL, Centrifuge, Conical</td>
			<td>Genesee Scientific</td>
			<td>28-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, Microcentrifuge, 1.5/2 mL</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Fisher Scientific</td>
			<td>01-912-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Beakers (50mL &#8211; 2L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge with cooling</td>
			<td>Fisher Scientific</td>
			<td>13-690-006</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging-bucket centrifuge with cooling</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>50-728-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Sonicator</td>
			<td>Fisher Scientific</td>
			<td>15-336-120</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of histone from sorghum leaf tissue for top down mass spectrometry profiling of potential epigenetic markers

石质属于真核生物中高度保存的蛋白质家族。他们把DNA包装成核糖体作为染色质的功能单位。分子的转化后修改（PTM）具有高度的动态性，可以通过酶添加或去除，在调节基因表达方面发挥着关键作用。在植物中，表观遗传因素，包括层石PTM，与它们对环境的适应反应有关。了解表观遗传控制的分子机制可以为创新的生物工程解决方案带来前所未有的机遇。在这里，我们描述了一个从高梁叶组织中分离核和纯化组织色调的协议。提取的组织结石可以通过自上而下的质谱（MS）加上在线反相（RP）液体色谱（LC）以完整的形式进行分析。可以很容易地识别同一组蛋白上多个 PTM 的组合和形态学。此外，还可使用自上而下的 LC-MS 工作流检测石材尾部剪报，从而产生核心结石（H4、H2A、H2B、H3）的全球 PTM 轮廓。我们以前曾将此协议应用于从大规模实地研究中收集的高梁叶组织中分析平石 PTM，旨在识别抗旱的表观遗传标记。该方案有可能被调整和优化为色素免疫沉淀测序（ChIP-seq），或研究类似植物中的平石PTM。

按照协议，可以通过LC-MS分析提取和识别组织音。原始数据和处理结果可在 MassIVE （https://massive.ucsd.edu/） 通过加入获得： MSV000085770。根据来自代表性样本的 TopPIC 结果（也可从 MassIVE 获得），我们确定了 303 个组蛋白蛋白（106 H2A、72 H2B、103 H3 和 22 个 H4 蛋白石）。还检测到共同纯化的核糖体蛋白形成物，通常在LC早期排出。它们通常由已识别的蛋白形成物的 20% 组成，但不会与 LC 梯度后期的组蛋?...

Watch this Scientific Journal Video about Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers at JoVE.com

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

该方案的制定是为了有效地从高梁叶材料中提取完整的组织结石，用于分析结石转化后的修饰，这些修饰可以作为潜在的表观遗传标记，以帮助工程抗旱作物。

从高梁叶组织中分离出希石，用于自上而下质量光谱分析潜在的表观遗传标记

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

我们的协议揭示了高梁植物发育和抗旱的表观遗传标记。这种分子理解将有助于开发更好的解决方案，使作物适应未来的极端气候。此协议从高梁叶组织中生成高纯度组织，适用于质谱仪对转化后修饰进行无针对性的分析。演示程序的将是沙丹·阿卜达利和塔妮娅·温克勒，来自EMSL的学士后研究助理。首先，用液氮研磨几片高梁叶，在零下80摄氏度的50毫升离心管中储存。使用大约四克叶粉对每个样品进行石块分析。准备文本手稿中描述的提取缓冲区 1、2A 和 2B，并添加蛋白酶抑制剂片剂以提取缓冲液 1，最终浓度为 0.2 倍。然后在地叶粉末中加入20毫升的预制萃取缓冲液，轻轻混合或漩涡10分钟。使用网格 100，每次用两毫升提取缓冲液冲洗过滤材料两次。接下来，将过滤液在3000倍G下，在4摄氏度的摆动桶转子中搅动10分钟，以颗粒状细胞碎片和大型亚细胞器。同时，通过将蛋白酶抑制剂添加到0.4倍的最终浓度中，准备提取缓冲区2A。丢弃超强剂，而不会干扰颗粒。然后将颗粒重新悬在预制提取缓冲区2A的5毫升中。在冰上孵化10分钟，轻轻混合。在 2，100 倍 G 下将溶液在 4 摄氏度下在摆动的桶转子中搅动 15 分钟，以颗粒碎屑和核。小心地去除超细颗粒，而不会干扰颗粒。通过将蛋白酶抑制剂添加到1X的最终浓度中，准备提取缓冲区2B。在离心后获得的颗粒中加入五毫升这种制备缓冲液。离心机在2，100倍G为15分钟，在4摄氏度的摆动桶转子到颗粒碎片和核。同时，通过添加蛋白酶抑制剂片剂来准备核裂解缓冲。小心地在不干扰颗粒的情况下倒出超纳坦，并将颗粒重新悬浮在裂解缓冲器的250微升中。以最大速度将溶液调高 15 秒，使其均匀化并重新悬索材料。然后在摄氏四度下声化五分钟，储存在零下八十摄氏度。解冻冷冻核样品，加入750微升5%瓜尼丁缓冲区。然后在摄氏四度下声化15分钟。将样品转移到一个两毫升的管子中，在摄氏四度下以10，000倍G旋转10分钟。同时，用两毫升丙酮和四毫升水冲洗离子交换色谱柱，以尽量减少表面的污染。将大约 200 到 300 微升的弱 cation 交换树脂加载到色谱柱上，让树脂沉淀下来。用瓜尼丁缓冲器清洗树脂四次，将管子和柱子放在冰上。将柱子放在一个两毫升的收集管上，将预制样品中的超纳坦慢慢加载到树脂床上，而不会干扰树脂。当溶液流过时，将凹痕加载回柱的顶部六到八次，以便最大限度绑定到树脂上。然后加载两毫升的5%瓜尼丁缓冲区，以洗掉柱子上的非石蛋白。用一毫升5%的瓜尼丁缓冲区将石蛋白埃卢特，并收集出这种蛋白。用 500 微升洗涤溶剂清洁三千达顿截断旋转过滤器，然后使用预先清洁的自旋过滤器脱盐剩余的溶剂。在LC-MS功能图的特定区域观察到主要的石蛋白，这表明实验的成功。这张有代表性的地图在破折号的箱子里显示了完整的全长石质。H2B 和 16 千达顿 H2A 蛋白型的 LC-MS 功能图显示，两者都有多个具有类似序列的同源词，如唯一的产品会话数所示。可以看到另一组没有长尾的H2A组音。通过检查此处显示的 LC-MS 特征图的质量差异，可以观察到 H4 平石蛋白中的 N 端乙酰化、附加赖氨酸乙酰化和甲基氨酸氧化。有两个蛋白质序列确定为H3石蛋白，H3.3和H3.2。电子转移分离碎片用于生成已识别的H3.2蛋白形的碎片光谱。前体离子和前一个和下一个 Ms1 光谱在这里显示，其匹配的同位素峰值以紫色突出显示。可使用序列覆盖图本地化后转换。通过比较蛋白形成体的相对丰度，发现了与样品条件特有的截断的石蛋白表的变化。仅在第三周和第九周对一些样本进行了H4的C端截断。对于H3.2，N端截断蛋白形成通常在第10周更为丰富。相比之下，C 端截断 H3.2 出现在较早的时间点。H4 C端截断的蛋白形成在BTx642中比在RTx430中更为丰富。在尝试此协议时，请密切注意超纳坦和颗粒的颜色。颜色变化有助于识别叶绿体研磨或裂解中的潜在问题。我们希望，这种从高梁叶中分离出层石的稳健方案将促进其他类似植物的表观遗传学研究。

Research

JoVE Journal

Biochemistry

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

Sheathless Capillary ElectrophoresisÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry for Metabolic Profiling of Biological Samples

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

叶喷雾质谱: 一种快速的环境电离技术直接评价植物组织中的代谢产物

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...