Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析
Summary
在这里, 我们提出了一个协议, 执行 polysome 分析的孤立灌注小鼠心脏。我们描述的方法, 心脏灌注, polysome 分析, 并分析 polysome 分数的基因, miRNAs, polysome 蛋白质组。
Abstract
蛋白质翻译中动态变化的研究需要专门的方法。在这里, 我们研究了新合成的蛋白质对缺血和再灌注反应的变化, 采用离体灌注小鼠心脏结合 polysome 分析。为了进一步了解蛋白质转化过程中的动态变化, 我们以胞内核糖体 (核糖体或 polysomes) 为基因, 并恢复了线粒体 polysomes, 并对其 mRNA 和蛋白质分布进行了比较。高效率的分数 (许多核糖体附着于 mRNA), 效率低下 (附着的核糖体较少), 也包括线粒体 polysomes 和非翻译分数。miRNAs 还可以与正在翻译的基因关联, 从而降低了翻译的效率, 我们研究了跨分数的 miRNAs 分布。对基因、miRNAs 和蛋白质在基础灌注条件下的分布进行了考察, 在30分钟的全球无流缺血和30分钟后再灌注。在这里, 我们介绍了用于完成这一分析的方法, 特别是从蔗糖梯度提取蛋白质的方法, 因为这一点以前没有描述过, 并提供了一些具有代表性的结果。
Introduction
心脏以动态的方式对缺血 (I) 和再灌注 (R) 的损伤进行反应。然而, 在反应过程中, 对蛋白质合成的急性变化没有什么洞察力。为了解决这一问题, 我们利用 polysome 分析1的成熟方法来确定蛋白质丰度的变化, 反映核糖体和转化调节因子从细胞质到 polysomes 的再分配, 以及增加新合成的蛋白质 (NSPs)。在 I/R 的设置中, 新蛋白合成的增加发生在与新基因2的转录不一致的时间范围内;此外, 在 mRNA 表达水平和蛋白质丰度之间的不一致报告了3。基于这些原因, 我们选择分析蛋白质翻译所反映的动态蛋白质组的变化。为了做到这一点, 我们量化的 polysome 分数 mRNA, 并分析蛋白质组成的 polysome 分数。最后, 由于 microRNAs (大鹏湾) 调节基因的可用性, 并能干扰蛋白质翻译4、5的效率, 我们研究了大鹏湾在 polysome 馏分中的分布情况, 着重于对我/R 的响应。
我们选择使用孤立的小鼠 Langendorff 灌注模型和收获组织在基础条件下连续灌注, 30 分钟的全球无流性缺血后, 30 分钟的缺血后30分钟再灌注。然后, 我们可溶性的心脏组织和分离的 polysomes 在蔗糖梯度, 其次是蛋白质组分析和选择性检测基因和 miRNAs 的 PCR 和微阵列, 分别。这种方法的组合代表了一个强大的方法来了解动态蛋白质组, 使同时检测 mRNA, miRNA 和 NSPs, 以及调节蛋白, miRNA 和 mRNA 之间的 nontranslating 分数之间的重新分配,低效率的 polysomes 和高效的 polysomes (见图 1)。通过进一步分析诸如 eIF2α或 mTOR 等关键调控因素的磷酸化, 可以进一步深入研究这一过程的动态调节。现在详细描述了这些单独的步骤。
Protocol
所有动物研究都是按照机构指南进行的, 并由雪松-西奈医学中心的机构动物护理和使用委员会批准。 1. Langendorff 灌注小鼠心脏 小鼠心脏缺血再灌注 Langendorff 灌注的研究 管理腹腔戊巴比妥钠70毫克/千克的成年小鼠 (8 周岁, 男性, C57BL6/j)。确认深部麻醉, 缺乏撤退到脚趾捏。 Anticoagulate 腹腔肝素 500 U/千克。 通过胸骨切口打开胸腔。?…
Representative Results
mRNA 分析mrna 的结果可以表达为一个特定的 mRNA 在每一个分数的分布 (图 3A);为量化, 结合 polyribosomal 翻译分数和比较非翻译分数 (图 3B), 提出了 mRNA 丰度在翻译到 nontranslating 分数的比率。额外的信息是通过检查高效率的 polysome 分数分别从低效率 polysome 分数 (并与 nontranslating 分数分开) 获得。这在分析 miRNAs 时尤为重要, …
Discussion
通过分析特定 mRNA 或整个转录6、7的平移状态, Polysome 剖面分析允许对蛋白质的翻译进行研究。当需要对本地翻译进行研究时, 如突触体8, 也有很大的帮助。传统上, 这种方法包括分离单和核糖体和相关的基因的蔗糖梯度, 可以结合基因组或蛋白质蛋白技术, 以获得预期结果6,9。例如, 我们小组?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
nih P01 HL112730 (抹布, JVE), nih R01 HL132075 (抹布, JVE), 芭芭拉史翠珊妇女心脏中心 (抹布, JVE), 桃乐茜和 e. 菲利普在分子心脏病学 (抹布) 的里昂椅子, 艾丽丝在妇女的心脏健康 (JVE) 和捷克科学院的椅子机构支持 RVO: 68081715 (MS)。
Materials
| Pentobarbital | Vortech Phamaceuticals | 9373 | for euthansia |
| Heparin | Sagent | 103424 | used in langendorff preparation |
| forceps | Fine Science Tools | 91110-10 | used to hang the heart |
| Langendorff system | Radnoti + home made | n/a | A 'four heart' system consisting of custom blown glass, tubing and water baths |
| NaCl | Sigma | S7653-5KG | Krebs buffer and Sucrose gradient |
| KCl | Sigma | P5405 | Krebs buffer and Lysis buffer |
| KH2PO4 | Sigma | P-5504 | Krebs buffer |
| MgSO4 | Sigma | M7774-500G | Krebs buffer |
| Glucose | Sigma | G5767 | Krebs buffer |
| CaCl2 | Sigma | C1016-500G | Krebs buffer |
| Sucrose powder | Sigma | S0389-1KG | Sucrose gradient |
| MgCl2 | Sigma | 208337 | Sucrose gradient and Lysis buffer |
| Tris-base | Sigma | T1503-1KG | Sucrose gradient and Lysis buffer |
| Xylene Cyanole | Sigma | X-4126 | Sucrose gradient |
| Cycloheximide | Sigma-aldrich | 239763 | Sucrose gradient and Lysis buffer |
| RNaseOUT | Life Technologies | C00019 | RNAse inhibitor for Lysis buffer |
| Igepal CA-360 (NP40) | Sigma | I3021 | Lysis buffer |
| Protease Inhibitor Cocktail tablets, EDTA free | Roche | 5056489001 | |
| Tube, Thinwall, Ultra-Clear, 13.2 mL, 14 x 89 mm | Beckman Coulter | 344059 | |
| Ultracentrifuge | Beckman | LE-80K | Ultracentrifugation of the gradients |
| Rotor | Beckman | SW41 | Ultracentrifugation of the gradients |
| Biologic LP (pump) | Biorad | 731-8300 | Fractionation of the gradients |
| BioFrac | Biorad | 741-0002 | Fractionation of the gradients |
| Eppendorf RNA/DNA LoBind microcentrifuge tubes, 2 mL tube | Sigma | Z666513-100EA | Gradient fraction and RNA extraction |
| TRIzol Reagent | Life technologies | AM9738 | RNA extraction |
| Luciferase Control RNA | Promega | L4561 | RNA extraction |
| Chloroform | Fisher Scientific | C606-4 | RNA extraction |
| Glycogen, RNA grade | Thermo Fisher Scientific | R0551 | RNA extraction |
| Isopropanol | Sigma | I9516 | RNA extraction |
| Ethanol | Sigma | E7023-1L | RNA extraction |
| iScript cDNA Synthesis Kit | BioRad | 170-8891 | Reverse transcription |
| iTaq Universal SYBR Green Supermix | BioRad | 175-5122 | Quantative PCR |
| miRNeasy Micro Kit (50) | Qiagen | 217084 | Kit for total RNA isolation |
| miScript II RT Kit (50) | Qiagen | 218161 | Kit for miRNA reverse transcription |
| miScript Sybr Green PCR Kit (200) | Qiagen | 218073 | Kit for real-time PCR expression analysis of miRNAs |
| Centrifuge 5424R | Eppendorf | For centrifugation of 1.5ml or 2.0ml tubes at different temperatures. Max speed – 21130g | |
| Centrifuge 5810R | Eppendorf | For real-time PCR plate centrifugation at different temperatures. Max speed – 2039g | |
| My Cycler Thermal Cycler | Bio-Rad | For reverse transcription | |
| CFX96 Real-Time System/C1000 Touch Thermal Cycler | Bio-Rad | For real-time PCR analysis | |
| miRNeasy Serum/Plasma Spike-in Control | Qiagen | 219610 | For quality control of RNA isolation |
| Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, green/clear | Bio-Rad | HSP9641 | For real-time PCR analysis |
| Microseal 'B' PCR Plate Sealing Film, adhesive, optical | Bio-Rad | MSB1001 | For real-time PCR plate sealing |
| Research plus Single-Channel Pipette, Gray; 0.5-10 µL | Eppendorf | UX-24505-02 | For pipetting |
| PIPETMAN Classic Pipets, P20 | Gilson | F123600G | For pipetting |
| PIPETMAN Classic Pipets, P200 | Gilson | F144565 | For pipetting |
| Rainin L-1000XLS Pipet-Lite XLS LTS Pipette 100-1000 µL | Gilson | 17011782 | For pipetting |
| Glycogen, RNA grade | Thermo Fisher Scientific | R0551 | Improves total RNA isolation efficiency |
| Posi-Click 1.7 mL Tubes, natural color | Denville | C2170 | RNA isolation and storage; reagent mix |
| Thermal Cycling Tubes -0.2 mL Individual Caps, Standard 0.2 mL tubes with optically | Denville | C18098-4 (1000910) | Reverse transcription reaction |
| Sharp 10 Precision barrier Tips | Denville | P1096-FR | For pipetting |
| Sharp 20 Precision barrier Tips | Denville | P1121 | For pipetting |
| Sharp 200 Precision barrier Tips | Denville | P1122 | For pipetting |
| Tips LTS 1 mL Filter | Rainin | RT-L1000F | For pipetting |
| miScript Primer Assay (200) | Qiagen | (it changes according to the miRNA) | For real-time PCR analysis |
| Gradient Master ver 5.3 Model 108 | BioComp Instruments | For preparation of sucrose gradients | |
| trichloroacetic acid | Sigma Aldrich | T6399 | |
| acetone | Sigma Aldrich | 650501 | |
| Tris hydrochloride | Amresco | M108 | |
| dithiothreitol | Fisher Scientific | BP172 | |
| iodoacetamide | Gbiosciences | RC-150 | |
| sequencing grade modified trypsine, porcine | Promega | V5111 | |
| ammonium bicarbonate | BDH | BDH9206 | |
| formic acid, Optima LC/MS | Fisher Chemical | A117 | |
| methanol, Optima LC/MS | Fisher Chemical | A454 | |
| acetonitrile, Optima LC/MS | Fisher Chemical | A996 | |
| Protein LoBind tubes 0.5 mL | Eppendorf AG | 22431064 | |
| Protein LoBind tubes 1.5 mL | Eppendorf AG | 22431081 | |
| HLB µElution plate 30 µm | Oasis | 186001828BA | |
| SpeedVac concentrator | Thermo Scientific | Savant SPD2010 | |
| sonicator | Qsonica | Oasis180 | |
| centrifuge | Thermo Scientific | Sorvall Legend micro 21R | |
| LC trap column PepMap 100 C18 | Thermo Scientific | 160454 | |
| LC separation column PepMap RSLC C18 | Thermo Scientific | 164536 | |
| mass spectrometer | Thermo Scientific | Orbitrap Elite ion trap mass spectrometer | |
| MSConvert software | ProteoWizard Toolkit | ||
| Sorcerer-SEQUEST software | Sage-N Research, Inc. | ||
References
- Pourpirali, S., Valacca, C., Merlo, P., Rizza, S., D’Amico, S., Cecconi, F. Prolonged pseudohypoxia targets ambra1 mrna to p-bodies for translational repression. PLoS ONE. 10, e0129750 (2015).
- Andres, A. M., Tucker, K. C., Thomas, A., Taylor, D. J., Sengstock, D., Jahania, S. M., Dabir, R., Pourpirali, S., Brown, J. A., Westbrook, D. G., Ballinger, S. W., Mentzer, R. M., Gottlieb, R. A. Mitophagy and mitochondrial biogenesis in atrial tissue of patients undergoing heart surgery with cardiopulmonary bypass. JCI Insight. 2, e89303 (2017).
- Kislinger, T., Cox, B., Kannan, A., Chung, C., Hu, P., Ignatchenko, A., Scott, M. S., Gramolini, A. O., Morris, Q., Hallett, M. T., Rossant, J., Hughes, T. R., Frey, B., Emili, A. Global survey of organ and organelle protein expression in mouse: Combined proteomic and transcriptomic profiling. Cell. 125, 173-186 (2006).
- Kren, B. T., Wong, P. Y., Shiota, A., Zhang, X., Zeng, Y., Steer, C. J. Polysome trafficking of transcripts and micrornas in regenerating liver after partial hepatectomy. American Journal of Physiology. Gastrointestinal and Liver Physiology. 297, G1181-G1192 (2009).
- Gottlieb, R. A., Pourpirali, S. Lost in translation: Mirnas and mrnas in ischemic preconditioning and ischemia/reperfusion injury. Journal of Molecular and Cellular Cardiology. 95, 70-77 (2016).
- Coudert, L., Adjibade, P., Mazroui, R. Analysis of translation initiation during stress conditions by polysome profiling. Journal of Visualized Experiments. , (2014).
- Lorsch, J. Methods in enzymology. Laboratory methods in enzymology: Rna. Preface. Methods in Enzymology. 530, xxi (2013).
- Kuzniewska, B., Chojnacka, M., Milek, J., Dziembowska, M. Preparation of polysomal fractions from mouse brain synaptoneurosomes and analysis of polysomal-bound mrnas. Journal of Neuroscience Methods. 293, 226-233 (2018).
- He, S. L., Green, R. Polysome analysis of mammalian cells. Methods in Enzymology. 530, 183-192 (2013).
- Molotski, N., Soen, Y. Differential association of micrornas with polysomes reflects distinct strengths of interactions with their mrna targets. RNA. 18, 1612-1623 (2012).
- Maroney, P. A., Yu, Y., Fisher, J., Nilsen, T. W. Evidence that micrornas are associated with translating messenger rnas in human cells. Nature Structural and Molecular Biology. 13, 1102-1107 (2006).
- Paul, P., Chakraborty, A., Sarkar, D., Langthasa, M., Rahman, M., Bari, M., Singha, R. S., Malakar, A. K., Chakraborty, S. Interplay between mirnas and human diseases. Journal of Cellular Physiology. 233, 2007-2018 (2018).
- Rupaimoole, R., Slack, F. J. Microrna therapeutics: Towards a new era for the management of cancer and other diseases. Nature Reviews Drug Discovery. 16, 203-222 (2017).
- Nelson, P. T., Hatzigeorgiou, A. G., Mourelatos, Z. Mirnp:Mrna association in polyribosomes in a human neuronal cell line. RNA. 10, 387-394 (2004).
- Nottrott, S., Simard, M. J., Richter, J. D. Human let-7a mirna blocks protein production on actively translating polyribosomes. Nature Structural and Molecular Biology. 13, 1108-1114 (2006).
- Kim, J., Krichevsky, A., Grad, Y., Hayes, G. D., Kosik, K. S., Church, G. M., Ruvkun, G. Identification of many micrornas that copurify with polyribosomes in mammalian neurons. Proceedings of the National Academy of Sciences of the United States of America. , 360-365 (2004).
- Androsavich, J. R., Chau, B. N., Bhat, B., Linsley, P. S., Walter, N. G. Disease-linked microrna-21 exhibits drastically reduced mrna binding and silencing activity in healthy mouse liver. RNA. 18, 1510-1526 (2012).
- Kraushar, M. L., Thompson, K., Wijeratne, H. R., Viljetic, B., Sakers, K., Marson, J. W., Kontoyiannis, D. L., Buyske, S., Hart, R. P., Rasin, M. R. Temporally defined neocortical translation and polysome assembly are determined by the rna-binding protein hu antigen r. Proceedings of the National Academy of Sciences of the United States of America. , E3815-E3824 (2014).
- McClatchy, D. B., Ma, Y., Liu, C., Stein, B. D., Martinez-Bartolome, S., Vasquez, D., Hellberg, K., Shaw, R. J., Yates, J. R. Pulsed azidohomoalanine labeling in mammals (palm) detects changes in liver-specific lkb1 knockout mice. Journal of Proteome Research. 14, 4815-4822 (2015).
- Schiapparelli, L. M., McClatchy, D. B., Liu, H. H., Sharma, P., Yates, J. R., Cline, H. T. Direct detection of biotinylated proteins by mass spectrometry. Journal of Proteome Research. 13, 3966-3978 (2014).
- Wiśniewski, J. R., Zougman, A., Nagaraj, N., Mann, M. Universal sample preparation method for proteome analysis. Nature Methods. 6, 359-362 (2009).
- Rajalingam, D., Loftis, C., Xu, J. J., Kumar, T. K. Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate. Protein Science. 18, 980-993 (2009).
- Fic, E., Kedracka-Krok, S., Jankowska, U., Pirog, A., Dziedzicka-Wasylewska, M. Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis. Electrophoresis. 31, 3573-3579 (2010).
- Sashital, D. G., Greeman, C. A., Lyumkis, D., Potter, C. S., Carragher, B., Williamson, J. R. A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30s subunit assembly in e. Coli. Elife. 3, (2014).
- Liang, S., Bellato, H. M., Lorent, J., Lupinacci, F. C. S., Oertlin, C., van Hoef, V., Andrade, V. P., Roffé, M., Masvidal, L., Hajj, G. N. M., Larsson, O. Polysome-profiling in small tissue samples. Nucleic Acids Research. 46, e3 (2018).
Cite This Article
Stastna, M., Thomas, A., Germano, J., Pourpirali, S., Van Eyk, J. E., Gottlieb, R. A. Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion. J. Vis. Exp. (138), e58079, doi:10.3791/58079 (2018).