二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组
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, 4The State Key Laboratory of Medical Genetics,Central South University
Summary
在这里, 我们提出了一个二维凝胶电泳 (2DE) 与质谱 (MS) 分离和鉴定人垂体腺瘤组织蛋白质组, 这是一个良好的和可重复的2DE 模式。在使用高灵敏度 MS 分析复杂的肿瘤蛋白质组时, 每2DE 点都能观察到许多蛋白质。
Abstract
人垂体腺瘤 (PA) 是一种常见的肿瘤, 发生在人垂体腺在下丘脑-垂体靶器官轴系统, 并可分为临床功能或非功能性 PA (平安险和消防协会)。由于与平安险相比, 在血液中几乎没有升高激素, 因此, 消防协会很难早期诊断和治疗。我们的长期目标是使用蛋白质组学方法发现可靠的生物标志物, 以澄清 PA 分子机制和识别有效的诊断, 预后标志和治疗目标。本文介绍了一种有效的二维凝胶电泳 (2DE) 结合质谱 (MS) 方法对人体 PA 蛋白质组进行分析, 包括制备样品、2D 凝胶电泳、蛋白质可视化、图像分析、凝胶胰蛋白酶消化, 肽质指纹图谱 (PMF) 和串联质谱 (ms/毫秒)。2维凝胶电泳基质辅助激光解吸/电离质谱 PMF (2 MALDI PMF), 2 MALDI ms/毫秒, 2 de 液相色谱 (LC) ms 和 ms 程序已成功地应用于对消防协会蛋白质组的分析。利用高灵敏度质谱仪, 在对复杂 PA 组织的分析中, 在每个2D 凝胶点上都发现了许多蛋白质, 并采用2的 LC-ms/毫秒方法, 以最大限度地提高人类 PA 蛋白质组的覆盖率。
Introduction
PA 是一种常见的肿瘤, 发生在人类垂体腺的下丘脑-垂体靶向系统, 这在人类内分泌系统中扮演重要的角色。PA 包括临床功能和非功能性 PAs (平安险和消防协会)1,2。在早期诊断和治疗方面, 消防协会是困难的, 因为只有轻微升高的荷尔蒙水平 (例如, LH, 和 FSH) 的血液比平安险, 这大大提高了血液中相应荷尔蒙的水平3,4 ,5。分子机制的澄清和有效生物标志物的发现对消防协会的诊断、治疗和预后具有重要的临床意义。我们的长期目标是开发和使用蛋白质组学方法研究消防协会, 以发现可靠的生物标志物, 以澄清其分子机制, 并确认有效的治疗目标, 以及诊断和预后标志。2-DE 结合 MS 方法已广泛应用于我们的长期研究计划, 关于人类 PA 蛋白质组1,2,6,7, 包括建立蛋白质组参考映射3,8, 差异表达的蛋白质剖面分析9,10,11,12,13, 荷尔蒙变体14 ,15, 平移后修改, 如磷酸化14和酪氨酸硝化16,17,18, 入侵相对的蛋白质组变异无创 NFPAs19和消防协会亚型13的蛋白质组异质性, 导致了多种重要通路网络 (线粒体功能障碍、细胞周期失调、氧化应激和 MAPK 信号) 的发现。系统异常) 在消防协会13,19,20中更改。
2DE. 根据它们的等电点 (pI) (电聚焦, IEF) 和分子量 (通过月桂酸钠聚丙烯酰胺凝胶电泳, SDS 页)1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23. 这是蛋白质组学领域的一种常见的和经典的分离技术, 自从 1995年24中引入了蛋白质组和蛋白质的概念。ms 是找出2种分离蛋白的关键技术, 包括 PMF 和 ms/ms 策略。MS 仪器的飞速发展, 特别是在检测灵敏度和分辨率方面, 结合 LC 系统的改进, 大大提高了蛋白质组中低或极低丰度蛋白质的识别率, 最大限度地蛋白质组的覆盖范围。它还挑战我们的传统观念, 在分析复杂的人体组织蛋白质组中只有一个或两个蛋白质存在于一个2D 凝胶点, 并提供了一个机会, 以确定在一个2D 凝胶点的多蛋白在复杂的人体组织分析蛋白质组, 并最大限度地覆盖了消防协会蛋白质组。
在这里, 我们描述了 2 MALDI ms PMF、2 MALDI ms/毫秒和2个非 LC ms/毫秒的详细协议, 这些方法已成功地用于人体消防协会蛋白质组的分析。这些协议包括: 样品的制备, 第一维 (电聚焦, IEF), 第二维 (SDS 页), 蛋白质的可视化 (银染色和考马斯蓝染色), 2D 凝胶的图像分析, 凝胶胰蛋白酶消化,纯化胰蛋白酶肽、PMF、ms/ms 和数据库灼热的3,8,25,26。此外, 该协议容易转化为分析其他人体组织蛋白质组。
Protocol
本议定书遵循中国中南大学象牙医院医学伦理学委员会的指导方针。在整个实验过程中应佩戴头帽和手套, 以避免皮肤和毛发中的角蛋白污染8。 1. 样品的制备 从神经外科部门收集 PA 组织 (0.2-0.5 毫克)。立即冷冻液氮, 然后转移到-80 °c 存放。 在去离子蒸馏水中加入2毫升0.9% 氯化钠 (ddH2O)。使用此解决方案可以轻松地从组织表面 (3x) 中?…
Representative Results
1. 2 MALDI MS PMF:使用上面描述的实验过程, 从 FSH 表达的消防协会组织 (女性; 50 岁, 促肾上腺皮质激素 (-), 生长激素 (-), 泌乳素 (-), LH (-), FSH (+) 和 TSH (-)) 中提取150µg 蛋白, 并排列18厘米IPG 条 (pH 3-10 NL) 和一个大格式的 SDS 页凝胶, 然后可视化银染色。我们获得了一个可重现和良好的2DE 凝胶模式的消防协会组织蛋白质组 (图 1), 平?…
Discussion
2DE, 再加上 ms 方法, 包括 2 PMF ms 和2的 ms/毫秒, 已成功地应用于我们的长期计划-使用蛋白质组学研究人类的消防协会蛋白质的变化和分子网络的变化, 以阐明分子机制和NFPAs 的有效生物标志物的发现。2基于 de 的比较蛋白质组学具有良好的重现性, 在鉴定消防协会蛋白质多样性方面起着重要作用9,10,11,12<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到中国国家自然科学基金 (81572278 和81272798对 xz) 的支持, 中国 “863” 计划项目 (2014AA020610-1)、象牙医院人才引进基金 (xz) 和湖南中国省级自然科学基金 (授予14JJ7008 号)。作者还承认 Desiderio 博士在田纳西大学健康科学中心的科学贡献。X.Z. 连续开发和使用2种方法从2001年起对垂体腺瘤蛋白质组进行分析, 构思了本手稿的概念, 撰写和修订了手稿, 协调了相关工作, 并负责财务支持及相应工作。Y.L. 参加了手稿的修订。他参加了参考资料的收集和手稿的修订。所有作者都批准了最后的手稿。
Materials
| Ettan IPGphor 3 | GE Healthcare | isoelectric focusing system. | |
| Ettan DALTsix multigel caster | Amersham Pharmacia Biotech, Piscataway, NJ, USA | ||
| Ettan DALT II System | Amersham Pharmacia Biotech, Piscataway, NJ, USA | The vertical electrophoresis system | |
| Ettan IPGphor strip holder | Amersham Pharmacia Biotech, Piscataway, NJ, USA | ||
| Ettan DALTsix multigel caster | Amersham Pharmacia Biotech, Piscataway, NJ, USA | Multigel caster | |
| Voyager DE STR MALDI-TOF MS | ABI, Foster City,CA | MALDI-MS PMF | |
| MALDI-TOF-TOF | Autoflex III, Bruker | MALDI-MS/MS mass spectrometer | |
| LTQ-OrbiTrap Velos Pro ETD | Thermo Scientific, Waltham, MA, USA | ESI-MS/MS mass spectrometer | |
| EASY-nano LC system | Proxeon Biosystems, Odense, Denmark | High performance liquid chromatography system | |
| PepMap C18 trap column | 300 μm i.d. × 5 mm length; Dionex Corp., Sunnyvale, CA, USA | ||
| RP C18 column | 75 μm i.d., 15 cm length; Dionex Corp., Sunnyvale, CA, USA | ||
| KimWipe | Kimvipe | Insoluble paper towel | |
| Watter | Made by PURELAB flex instrument | ||
| Polytron Model P710/35 homogenizer | Brinkmann Instruments, Westbury, NY | ||
| PDQuest | Bio-Rad, Hercules, CA | 2D gel image analysis software | |
| SEQUEST | Thermo Proteome Discoverer 1.3 (version No. 1.3.0.339) | ||
| DataExplore (ver. 4.0.0.0) software | MS spectrum-processing software | ||
| Mascot software | PMF-based protein searching software | ||
| Mascot software | MS/MS-based protein searching software | ||
| Proteome Discoverer software v.1.3 beta | Thermo Scientific | ||
| Xcalibur software v.2.1 | MS/MS data-acquired management software | ||
| Uniprot version 201410.1_HUMAN.fasta | Human protein database | ||
| SEQUEST (version No. 1.3.0.339) | MS/MS-based protein searching software I | ||
| MASCOT (version 2.3.02) | MS/MS-based protein searching software II | ||
| C18 ZipTip microcolumn | Millipore | ||
| PeptideMass Standard kit | Perspective Biosystems | ||
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23227 | |
| 2-D Quant Kit | GE Healthcare | 80-6483-56 | |
| BIS-ACRYLAMIDE | AMRESCO | 0172 | |
| ACRYLAMIDE | AMRESCO | 0341 | |
| DTT | Sigma-Aldrich | D0632 | |
| Thiourea | Sigma-Aldrich | T8656 | |
| Urea | VETEC | V900119 | |
| SDS | AMRESCO | 0227 | |
| CHAPS | AMRESCO | 0465 | |
| TEMED | AMRESCO | M146 | |
| Ammonium Persulfate | AMRESCO | M133 | |
| Trypsin | Promega, Madison, WI, USA | V5111 | |
| IPG buffer pH 3-10, NL | GE Healthcare | 17-6000-87 | |
| Immobiline Dry Strip pH 3-10NL,18cm | GE Healthcare | 17-1235-01 |
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Citer Cet Article
Zhan, X., Huang, Y., Long, Y. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome. J. Vis. Exp. (134), e56739, doi:10.3791/56739 (2018).