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
1Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital,Central South University, 2Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital,Central South University, 3State Local Joint Engineering Laboratory for Anticancer Drugs, Xiangya Hospital,Central South University, 4National Clinical Research Center for Geriatric Disorders, Xiangya Hospital,Central South University, 5Department of Obstetrics and Gynecology, Xiangya Hospital,Central South University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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?…
Representative Results
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?…
Discussion
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| BCA protein assay kit | Vazyme | E112 | 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). |
| Bovine serum albumin (BSA) | Solarbio | A8020-5G | Heat shock fraction, Australia origin, protease free, low fatty acid, low IgG, pH 7, ≥98% |
| Centrifuge | XiangYi | TDZ4–WS | |
| CHAPS | Sigma | C9426-5G | BioReagent, suitable for electrophoresis, ≥98% (HPLC) (Sigma-Aldrich) |
| Diamine tetraacetic acid (EDTA) | Sigma | 798681-100G | Anhydrous, free-flowing, Redi-Dri, ≥98% |
| DTT | Sigma | 10197777001 | 1,4-Dithiothreitol |
| Easy nLC | Proxeon Biosystems (now Thermo Fisher Scientific) | ||
| Ethylen glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA) | Sigma | E0396-10G | BioXtra, ≥97 .0% |
| Homogenizer | SilentShake | HYQ-3110 | |
| iTRAQ reagent kit | Applied Biosystems | Applied Biosystems iTRAQ Reagents–Chemistry Reference Guide, P/N 4351918A | |
| Low-temperature super-speed centrifuger | Eppendorf | 5424R | |
| Mannitol | Macklin | M813424-100G | 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. |
| MASCOT search engine | Matrix Science, London, UK; version 2.2 | ||
| Nagarse | Solarbio | P9090 | |
| N-hydroxysuccinimide (SDT) | Sigma | 56480-25G | Purum, ≥97.0% (T) |
| Nycodenz | Alere/Axis-Shield | 1002424-1 | |
| Phenylmethanesulfonyl fluoride (PMSF) protease inhibitor | Solarbio | P0100-1ML | PMSF is a protease inhibitor that reacts with serine residues to inhibit trypsin, chymotrypsin, thrombin, and papain. |
| Potassium chloride | Macklin | P816354-25G | Potassium chloride, KCI, also known as potassium muriate and sylvite, is a colorless crystalline solid with a salty taste that melts at 776°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. |
| Proteome Discover 1.4 | Matrix Science, London, UK | ||
| PVDF membrane | Millipore | 05317 | It is 1 roll, 26.5 cm x 1.875 m, 0.45 µm pore size, hydrophobic PVDF transfer membrane with low background fluorescence for western blotting. It is compatible with visible and infrared fluorescent probes. |
| Q Exactive mass spectrometer | Thermo Fisher Scientific | ||
| SCX column | Sigma | 58997 | It is 5-μm particle size, length 5cm × i.d. 4.6mm (Supelco). |
| Sodium orthovanadate (V) | Macklin | S817660-25G | 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. |
| Sucrose | Macklin | S824459-500G | Vetec reagent grade, 99% |
| Thiourea | Sigma | 62-56-6 | ACS reagent, ≥99.0% |
| Tris base | Sigma | 10708976001 | TRIS base is useful in the pH range of 7.0-9.0. It has a pKa of 8.1 at 25°C. |
| Trypsin (cell culture use) | Gibco | 25200-056 | 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. |
| Urea | Sigma | U5378-100G | powder, BioReagent, for molecular biology, suitable for cell culture |
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Citar este artículo
Zhan, X., Li, H., Qian, S., Zhan, X., Li, N. Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis. J. Vis. Exp. (153), e60435, doi:10.3791/60435 (2019).