Name: preparation of mitochondria from ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis
Uploaded: 2019-11-18T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis at JoVE.com

这项工作得到了湖南省百人计划（至X.Z.）、湘雅医院人才引进基金（XZ）、国家自然科学基金（第81572278号、81272798至XZ）的资助，中国"863"计划资助。项目（授权号2014A020610-1至XZ）和中国湖南省自然科学基金（批号14JJ7008至XZ）。X.Z. 构思了本稿件的概念，获取了 iTRAQ 定量蛋白质组学线样本的数据，编写和修订了手稿，协调了相关工作，并负责财务支持和相应的工作。H.L.制备线粒体样本。S.Q. 参与了部分工作。X.H.Z参与编写和编辑英语。N.L. 分析了 iTRAQ 蛋白质组学数据。所有作者都批准了最终的手稿。

该协议使用卵巢组织样本，包括卵巢癌组织（n = 7）和正常对照卵巢组织（n = 11）。本议定书3、4、5号方案经中南大学湘雅医院医学伦理委员会批准。
1. 从人类卵巢癌组织制备线粒体
<ol><li>通过混合 210 mM 甘醇、70 mM 蔗糖、100 mM 氯化钾 （KCl）、50 mM Tris-HCl、1 mM 二胺四乙酸 （EDTA）、0.1 mM 乙二醇二（2-氨?...

<ol>
	<li>Sakhuja, S., Yun, H., Pisu, M., Akinyemiju, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Availability+of+healthcare+resources+and+epithelial+ovarian+cancer+stage+of+diagnosis+and+mortality+among+Blacks+and+Whites.">Availability of healthcare resources and epithelial ovarian cancer stage of diagnosis and mortality among Blacks and Whites.</a> Journal of Ovarian Research. 10, 57 (2017).</li><li>Gadducci, 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=Surveillance+procedures+for+patients+treated+for+epithelial+ovarian+cancer:+a+review+of+the+literature.">Surveillance procedures for patients treated for epithelial ovarian cancer: a review of the literature.</a> International Journal of Gynecological Cancer. 17, 21-31 (2007).</li><li>Li, N., Li, H., Cao, L., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+mitochondrial+proteome+in+human+ovarian+carcinomas.">Quantitative analysis of the mitochondrial proteome in human ovarian carcinomas.</a> Endocrine-Related Cancer. 25, 909-931 (2018).</li><li>Li, N., Zhan, X., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lncRNA+SNHG3+regulates+energy+metabolism+of+ovarian+cancer+by+an+analysis+of+mitochondrial+proteomes.">The lncRNA SNHG3 regulates energy metabolism of ovarian cancer by an analysis of mitochondrial proteomes.</a> Gynecologic Oncology. 150, 343-354 (2018).</li><li>Li, N., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathway+network+alterations+in+human+ovarian+cancers+identified+with+quantitative+mitochondrial+proteomics.">Signaling pathway network alterations in human ovarian cancers identified with quantitative mitochondrial proteomics.</a> EPMA Journal. 10, 153-172 (2019).</li><li>Deng, P., Haynes, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+cancer:+Potential+roles+of+ATF5+and+the+mitochondrial+UPR.">Mitochondrial dysfunction in cancer: Potential roles of ATF5 and the mitochondrial UPR.</a> Semin Cancer Biology. 47, 43-49 (2017).</li><li>Georgieva, 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=Mitochondrial+dysfunction+and+redox+imbalance+as+a+diagnostic+marker+of+"free+radical+diseases".">Mitochondrial dysfunction and redox imbalance as a diagnostic marker of "free radical diseases".</a> Anticancer Research. 37, 5373-5381 (2017).</li><li>Sotgia, F., 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+mitochondrial+based+oncology+platform+for+targeting+cancer+stem+cells+(CSCs):+MITO-ONC-RX.">A mitochondrial based oncology platform for targeting cancer stem cells (CSCs): MITO-ONC-RX.</a> Cell Cycle. 17, 2091-2100 (2018).</li><li>Lee, J. H., 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=Individualized+metabolic+profiling+stratifies+pancreatic+and+biliary+tract+cancer:+a+useful+tool+for+innovative+screening+programs+and+predictive+strategies+in+healthcare.">Individualized metabolic profiling stratifies pancreatic and biliary tract cancer: a useful tool for innovative screening programs and predictive strategies in healthcare.</a> EPMA Journal. 9, 287-297 (2018).</li><li>Zhan, X., Long, Y., Lu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+variations+in+proteome+and+metabolome+for+predictive+diagnostics+and+personalized+treatment+algorithms:+Innovative+approach+and+examples+for+potential+clinical+application.">Exploration of variations in proteome and metabolome for predictive diagnostics and personalized treatment algorithms: Innovative approach and examples for potential clinical application.</a> Journal of Proteomics. 188, 30-40 (2018).</li><li>Lu, M., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crucial+role+of+multiomic+approach+in+cancer+research+and+clinically+relevant+outcomes.">The crucial role of multiomic approach in cancer research and clinically relevant outcomes.</a> EPMA Journal. 9, 77-102 (2018).</li><li>Hu, R., Wang, X., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-parameter+systematic+strategies+for+predictive,+preventive+and+personalised+medicine+in+cancer.">Multi-parameter systematic strategies for predictive, preventive and personalised medicine in cancer.</a> EPMA Journal. 4, 2 (2013).</li><li>Cheng, T., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+for+predictive,+preventive,+and+personalized+medicine+in+cancer.">Pattern recognition for predictive, preventive, and personalized medicine in cancer.</a> EPMA Journal. 8, 51-60 (2017).</li><li>Zhan, X., Zhou, T., Li, N., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differentially+mitochondrial+proteomic+dataset+in+human+ovarian+cancer+relative+to+control+tissues.">The differentially mitochondrial proteomic dataset in human ovarian cancer relative to control tissues.</a> Data in Brief. 20, 459-462 (2018).</li><li>McMillan, J. D., Eisenback, 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=Transmission+electron+microscopy+for+analysis+of+mitochondria+in+mouse+skeletal+muscle.">Transmission electron microscopy for analysis of mitochondria in mouse skeletal muscle.</a> Bio-protocol. 8, e2455 (2018).</li><li>Paul, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+ablation+of+TRAF6+inhibits+skeletal+muscle+wasting+in+mice.">Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice.</a> Journal of Cell Biology. 191, 1395-1411 (2010).</li><li>Pascual-Ahuir, A., Manzanares-Estreder, S., Proft, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-+and+antioxidant+functions+of+the+peroxisome-mitochondria+connection+and+its+impact+on+aging+and+disease.">Pro- and antioxidant functions of the peroxisome-mitochondria connection and its impact on aging and disease.</a> Oxidative Medicine and Cellular Longevity. 2017, 9860841 (2017).</li><li>Schrader, M., Costello, J., Godinho, L. F., Islinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome-mitochondria+interplay+and+disease.">Peroxisome-mitochondria interplay and disease.</a> Journal of Inherited Metabolic Disease. 38, 681-702 (2015).</li><li>Bartol&#225;k-Suki, E., Imsirovic, J., Nishibori, Y., Krishnan, R., Suki, B. <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+mitochondrial+structure+and+dynamics+by+the+cytoskeleton+and+mechanical+factors.">Regulation of mitochondrial structure and dynamics by the cytoskeleton and mechanical factors.</a> International Journal of Molecular Sciences. 18, 1812 (2017).</li><li>Rezaul, K., Wu, L., Mayya, V., Hwang, S., Han, 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+systematic+characterization+of+mitochondrial+proteome+from+human+T+leukemia+cells.">A systematic characterization of mitochondrial proteome from human T leukemia cells.</a> Molecular &amp; Cellular Proteomics. 4, 169-181 (2005).</li><li>Zhan, 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=How+many+proteins+can+be+identified+in+a+2DE+gel+spot+within+an+analysis+of+a+complex+human+cancer+tissue+proteome?.">How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?.</a> Electrophoresis. 39, 965-980 (2018).</li><li>Zhan, X., Li, N., Zhan, X., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revival+of+2DE-LC/MS+in+proteomics+and+its+potential+for+large-scale+study+of+human+proteoforms.">Revival of 2DE-LC/MS in proteomics and its potential for large-scale study of human proteoforms.</a> Med One. 3, e180008 (2018).</li></ol>

线粒体改变是卵巢癌的标志。从人类卵巢癌中制备高质量的线粒体样本，控制组织用于大规模定量蛋白质组学，有助于深入了解卵巢癌发病机制和线粒体分子网络变化中的线粒体功能，有助于阐明其分子机制，为后续发现基于线粒体4、5、8的目标疗法和有效的生物标志物。微分速度离心与密度梯度离心相结合，有效地从人类卵...

卵巢癌是一种常见的妇科癌症，死亡率高，但分子机制1，2不清楚。大多数卵巢癌在晚期被诊断，这严重阻碍了治疗。线粒体变化是人类卵巢癌的标志，线粒体是能量代谢、细胞信号和氧化应激3、4、5、6、7的中心。深入了解卵巢癌中线粒体蛋白酶与控制卵巢组织的变化，将有助于深入了解卵巢癌的分子机制，以及发现有效可靠的生物标志物和治疗靶点。线粒体代谢已被提出并确认为癌症治疗的目标，抗线粒体治疗最终可能非常有利于预防癌症的复发和转移。个体代谢分析也已经实践为癌症分层和预测策略的有用工具9，10。
这项研究的长期目标是开发和使用定量线粒体蛋白质组学方法来研究卵巢癌，以澄清卵巢癌与控制卵巢组织之间的线粒体蛋白质组变化，以及其分子网络从系统多组分角度11、12的变化，从而发现线粒体靶向分子生物标记物13，以澄清卵巢癌的分子机制，预测，并个性化治疗卵巢癌患者。相对和绝对定量（iTRAQ）标签3、4的等值标签是定量线粒体蛋白质变化的有效方法。从人类卵巢癌中制备高质量的线粒体样本和控制卵巢组织是iTRAQ定量分析线粒体蛋白酶3的先决条件。线粒体制剂与iTRAQ定量蛋白质组学已成功地用于有关人类卵巢癌线粒体蛋白质组的长期研究项目，包括建立线粒体蛋白质组参考图3，分析微分表达线粒体剖面4、14和翻译后修饰，包括磷酸化，这已经导致发现重要的信号通路网络变化在人类卵巢癌5，包括能量代谢的变化4，脂质代谢，和米托巴细胞通路系统3。
此前的研究发现，微差速离心与密度梯度离心相结合是从人类卵巢癌中分离和纯化线粒体，控制卵巢组织3、4、5、14的有效方法。iTRAQ 标签与强阳离子交换 （SCX） -液相色谱 （LC）-串联质谱法 （MS/MS） 相结合，是检测、识别和量化制备线粒体样本中的蛋白质的关键技术。
这里，描述了线粒体制备与iTRAQ定量蛋白质组学的详细方案。这些已经成功地用于分析人类卵巢癌组织线粒体。这些方案包括样品制备、差分速度离心、密度梯度离心、线粒体样品的质量评估、胰蛋白酶的蛋白质消化、iTRAQ标记、SCX分馏、LC、MS/MS、数据库搜索以及线粒体蛋白的定量分析。此外，该协议很容易翻译分析其他人体组织线粒体蛋白酶。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Vazyme</td>
			<td>E112</td>
			<td>BCA protein assay kit is a special 3-component version of our popular BCA reagents, optimized to measure (A562nm) total protein concentration of dilute protein solutions (0.5 to 20 micrograms/ml).</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Solarbio</td>
			<td>A8020-5G</td>
			<td>Heat shock fraction, Australia origin, protease free, low fatty acid, low IgG, pH 7, &#8805;98%</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>XiangYi</td>
			<td>TDZ4--WS</td>
			<td></td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma</td>
			<td>C9426-5G</td>
			<td>BioReagent, suitable for electrophoresis, &#8805;98% (HPLC) (Sigma-Aldrich)</td>
		</tr>
		<tr>
			<td>Diamine tetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>798681-100G</td>
			<td>Anhydrous, free-flowing, Redi-Dri, &#8805;98%</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>10197777001</td>
			<td>1,4-Dithiothreitol</td>
		</tr>
		<tr>
			<td>Easy nLC</td>
			<td>Proxeon Biosystems (now Thermo Fisher Scientific)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylen glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA)</td>
			<td>Sigma</td>
			<td>E0396-10G</td>
			<td>BioXtra, &#8805;97 .0%</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>SilentShake</td>
			<td>HYQ-3110</td>
			<td></td>
		</tr>
		<tr>
			<td>iTRAQ reagent kit</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Applied Biosystems iTRAQ Reagents&#8211;Chemistry Reference Guide, P/N 4351918A</td>
		</tr>
		<tr>
			<td>Low-temperature super-speed centrifuger</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Macklin</td>
			<td>M813424-100G</td>
			<td>Mannitol is a polyol (polyhydric alcohol) produced from hydrogenation from fructose that functions as a sweetener, humectant, and bulking agent. It has low hygroscopicity and poor oil solvency.</td>
		</tr>
		<tr>
			<td>MASCOT search engine</td>
			<td>Matrix Science, London, UK; version 2.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nagarse</td>
			<td>Solarbio</td>
			<td>P9090</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (SDT)</td>
			<td>Sigma</td>
			<td>56480-25G</td>
			<td>Purum, &#8805;97.0% (T)</td>
		</tr>
		<tr>
			<td>Nycodenz</td>
			<td>Alere/Axis-Shield</td>
			<td>1002424-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF) protease inhibitor</td>
			<td>Solarbio</td>
			<td>P0100-1ML</td>
			<td>PMSF is a protease inhibitor that reacts with serine residues to inhibit trypsin, chymotrypsin, thrombin, and papain.</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Macklin</td>
			<td>P816354-25G</td>
			<td>Potassium chloride, KCI, also known as potassium muriate and sylvite, is a colorless crystalline solid with a salty taste that melts at 776&#176;C (1420 OF). It is soluble in water, but insoluble in alcohol. Potassium chloride is used in fertilizers, pharmaceuticals, photography, and as a salt substitute.</td>
		</tr>
		<tr>
			<td>Proteome Discover 1.4</td>
			<td>Matrix Science, London, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>05317</td>
			<td>It is 1 roll, 26.5 cm x 1.875 m, 0.45 &#181;m pore size, hydrophobic PVDF transfer membrane with low background fluorescence for western blotting. It is compatible with visible and infrared fluorescent probes.</td>
		</tr>
		<tr>
			<td>Q Exactive mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SCX column</td>
			<td>Sigma</td>
			<td>58997</td>
			<td>It is 5-&#956;m particle size, length 5cm &#215; i.d. 4.6mm (Supelco).</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate (V)</td>
			<td>Macklin</td>
			<td>S817660-25G</td>
			<td>Sodium orthovanadate (Vanadate) is a general competitive inhibitor for protein phosphotyrosyl phosphatases. The inhibition by sodium orthovanadate is reversible upon the addition of EDTA or by dilution.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Macklin</td>
			<td>S824459-500G</td>
			<td>Vetec reagent grade, 99%</td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma</td>
			<td>62-56-6</td>
			<td>ACS reagent, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>10708976001</td>
			<td>TRIS base is useful in the pH range of 7.0-9.0. It has a pKa of 8.1 at 25&#176;C.</td>
		</tr>
		<tr>
			<td>Trypsin (cell culture use)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>This liquid formulation of trypsin contains EDTA and phenol red. Gibco Trypsin-EDTA is made from trypsin powder, an irradiated mixture of proteases derived from porcine pancreas. Due to its digestive strength, trypsin is widely used for cell dissociation, routine cell culture passaging, and primary tissue dissociation.</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma</td>
			<td>U5378-100G</td>
			<td>powder, BioReagent, for molecular biology, suitable for cell culture</td>
		</tr>
	</tbody>
</table>

preparation of mitochondria from ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis

卵巢癌是一种常见的妇科癌症，死亡率高，但分子机制不明确。大多数卵巢癌在晚期被诊断，这严重阻碍了治疗。线粒体变化是人类卵巢癌的标志，线粒体是能量代谢、细胞信号和氧化应激的中心。深入了解卵巢癌中线粒体蛋白酶与控制卵巢组织的变化，将有助于深入了解卵巢癌的分子机制，以及发现有效可靠的生物标志物和治疗靶点。这里介绍了一种有效的线粒体制备方法，结合相对和绝对定量 （iTRAQ） 定量蛋白质组学的等值标记，用于分析人类卵巢癌和控制线粒体蛋白酶， 包括差分速度离心、密度梯度离心、线粒体样品质量评估、蛋白消化与胰蛋白酶、iTRAQ 标签、强阳离子交换分馏 （SCX）、液相色谱 （LC）、串联质谱 （MS/MS），数据库分析和线粒体蛋白的定量分析。许多蛋白质已被成功识别，以最大限度地覆盖人类卵巢癌线粒体蛋白酶，并实现人类卵巢癌中不同表达的线粒体蛋白特征。

从卵巢癌组织和控制卵巢组织对线粒体的制备有差异。这项研究发现，从卵巢癌组织制备线粒体比从控制卵巢组织3，4容易得多。必须对从控制卵巢组织制备线粒体的方案进行一些改进。首先，在组织均质化之前，有必要在添加到切碎控制组织的PBS溶液中加入8 mL的0.05%胰蛋白酶/20 mM EDTA，随后在室温下消化30分钟，在200 x g?...

Watch this Scientific Journal Video about Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis at JoVE.com

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

本文提出了一种将微分速度离心与密度梯度离心相结合，将线粒体从人类卵巢癌组织中分离出来并控制卵巢组织的定量蛋白质组学分析方案，生成人类卵巢癌线粒体蛋白体的高通量和高可重复性定量蛋白质组学分析的高质量线粒体样本。

卵巢癌组织线粒体的制备与控制卵巢组织进行定量蛋白质组学分析

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

线粒体变化在人类卵巢癌中起着重要作用。该协议建立了一个有效的平台，从人类卵巢癌和控制组织分离和纯化线粒体，进行大规模蛋白质组学分析。线粒体，作为能量代谢、细胞信号和氧化应激的中心，对卵巢癌线粒体蛋白质组变化的洞察有助于我们理解分子机制，并且是有效生物标志物和治疗目标的发现。该技术使用差速离心与密度梯度离心相结合，将线粒体与控制组织中人类卵巢癌分离，从而获得高质量的线粒体样本，用于定量蛋白质组学分析。线粒体制剂，加上 iTRAQ 定量蛋白质组学，已成功用于人类卵巢癌组织分析，这很容易转化为分析其他组织，线粒体蛋白质组学。那些从来没有执行过这种方法的人可能会发现，与癌症组织一样，将线粒体从卵巢控制中分离出可能很困难。有必要添加 trypsin 来改善控制组织均质化，并添加额外的密度梯度介质层，以改善线粒体分离。要开始此过程，请准备 250 毫升的线粒体隔离缓冲器，如文本协议中所述。将大约 1.5 克卵巢癌组织放在干净的玻璃盘中。加入两毫升预冷却线粒体分离缓冲液，轻轻清洗组织表面的血液。重复此洗涤三次。然后，使用干净的眼科剪刀将组织完全切碎成大约一立方毫米的碎片，然后将切碎的组织转移到50毫升的离心管中。加入13.5毫升含有纳加塞的线粒体分离缓冲液，浓度为每毫升0.2毫克。使用电子均质器在四摄氏度下使切碎的组织均质两分钟。在此之后，再添加三毫升线粒体分离缓冲液到组织均质，通过移液混合。将准备好的组织在1，300 xg下离心，在4摄氏度下离心10分钟。取出颗粒并保留上一液。接下来，将上一代在10000 xg和4摄氏度下离心10分钟。取出含有微量的上一物质，并保留颗粒。加入2毫升线粒体隔离缓冲液，通过光移液彻底悬浮颗粒。在 7000 xg 和 4 摄氏度下将这种颗粒悬浮液离心 10 分钟。丢弃上一提液，并保留含有粗线粒体的颗粒。加入12毫升25%密度梯度介质，重新悬浮提取的粗线粒体。如文本协议中所述，制作包含重新悬浮粗线粒体的不连续密度梯度，并在 52，000 xg 和 4 摄氏度下离心 90 分钟。使用长而钝的注射器收集25%和30%梯度介质之间的界面上净化的线粒体，然后将其转移到干净的管子上。将线粒体隔离缓冲液添加到收集的线粒体中，将其稀释到三倍体积。在15，000 xg下离心，在4摄氏度下20分钟。丢弃上一液，并保留颗粒。收集含有纯化线粒的最终颗粒，并将其储存在20摄氏度。首先，准备250毫升的线粒体隔离缓冲液，如文本协议中所述。将大约 1.5 克的正常控制卵巢组织添加到干净的玻璃盘中。加入两毫升预冷却线粒体分离缓冲液，轻轻清洗组织表面的血液。重复此洗涤三次。使用干净的眼科剪刀，将组织完全切碎成大约 1 立方毫米的碎片，然后将切碎的组织转移到干净的 50 毫升管中。将含有0.05%trypsin和20毫摩尔EDTA和PBS的溶液的8毫升加入到切碎的控制组织中，并在室温下消化30分钟，帮助细胞酶和释放线粒体。在200 xg下离心5分钟。丢弃上一液，保留组织和细胞。加入13.5毫升含有纳加塞的线粒体分离缓冲液，浓度为每毫升0.2毫克，并使用电动均质器在4摄氏度下使切碎的组织均质两分钟。再将三毫升线粒体分离缓冲液加入组织均质，通过移液彻底混合。在1，300 xg和4摄氏度下将准备好的组织均质离心10分钟。取出颗粒并保留上一液。将上一代在10000xg和4摄氏度下离心10分钟。取出含有微体的上一液，并保留颗粒。然后，加入2毫升线粒体隔离缓冲液，通过轻移液彻底悬浮颗粒。在 7000 xg 和 4 摄氏度下将这种颗粒悬浮液离心 10 分钟。丢弃上一提液，并保留含有粗线粒体的颗粒。加入12毫升25%密度梯度介质，重新悬浮提取的粗线粒体。如文本协议中所述，制作包含粗线粒体的不连续密度梯度，并在 52，000 xg 和 4 摄氏度下离心 90 分钟。使用长而钝的注射器收集纯化线粒体，范围从 25 和 30% 梯度介质之间的接口到 34 和 38% 梯度介质之间的接口。将净化的线粒体转移到干净的管子中。将线粒体隔离缓冲液添加到收集的线粒体中，将其稀释到三倍体积。在15，000 xg和4摄氏度下离心20分钟，然后丢弃上一代。加入2毫升线粒体分离缓冲液，在15，000 xg和4摄氏度下重新悬浮颗粒和离心机20分钟。丢弃上一液并保留颗粒。收集最后的颗粒，并将其储存在20摄氏度。在这项研究中，从人类卵巢癌和控制组织中为大规模定量蛋白质组学准备了高质量的线粒体样本。在卵巢癌组织和控制卵巢组织进行线粒体的准备方面有一些差异，包括使用不同的不连续密度梯度。离心后，从卵巢癌组织的纯化线粒体在25%至30%的界面发现，而来自对照卵巢组织的线粒体在25%至30%的界面到34%至38%之间的界面范围内，然后通过电子显微镜通过差速离心和密度梯度离心评估准备好的线粒体的质量。电子显微镜图像表明，在卵巢癌和控制卵巢组织中，分离的主要细胞器是线粒体，除了少量的过氧化物体。然而，线粒体的形态在卵巢癌中的变化比控制卵巢组织的变化要大。准备好的线粒体的质量也通过西部印迹进行评估。西方的印迹图像还表明，从卵巢癌和控制卵巢中准备好的线粒体样本中的主要成分是线粒体，除了少量的过氧化物体，这与电子显微镜分析一致。将过氧化物包含在准备好的线粒体中是合理的，因为线粒体与过氧化物体广泛相互作用，这反过来又反映了线粒体的功能完整性。这些结果表明，已准备好的线粒体样品具有高质量。当我执行这个程序时，重要的是，你完全切碎组织之前均质化，并添加三叶辛到切碎的控制组织。同样重要的是，您为癌症样本和控件制作和/或准备不连续密度梯度。按照这个程序，iTRAQ或TMT定量蛋白质组或磷蛋白组可以执行，以澄清卵巢癌线粒体蛋白质组轮廓，和磷蛋白体配置文件可以建立，以澄清线粒体在卵巢癌在巨大深度的作用。该技术为研究人员系统地研究卵巢癌的线粒体蛋白质组和磷蛋白群，构建信号通路网络系统，发现可靠的生物标志物和治疗靶点铺平了道路。

Research

JoVE Journal

Cancer Research

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

科研

癌症研究

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues

人肺组织中ZW10相互作用蛋白免疫染色的数字化分析

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image ...

Characterization and Functional Prediction of Bacteria in Ovarian Tissues

卵巢组织细菌的特征和功能预测

The theory of a "sterile" female upper reproductive tract has been encountering increasing opposition due to advancements in bacterial detection. However, ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

使用单链DNA免疫荧光量化卵巢癌细胞中的复制应激

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity

AlDeSense在基于醛脱氢酶1A1活性的卵巢癌细胞分层中的应用

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

用于临床前药物测试的卵巢癌患者来源的类器官模型

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

Human Omentum Specimen Processing and Co-Culture with Ovarian Cancer Cells

人网膜标本处理和与卵巢癌细胞共培养

建立卵巢癌-网膜相互作用的 3D 离体模型

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

作者聚焦：用于研究癌症-脂肪微环境相互作用的高级离体模型 

基于人网膜的卵巢癌腹膜转移离 体 模型

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...