从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征
Summary
在这里，我们描述了使用通过在细胞培养物（SILAC）氨基酸稳定同位素标记的技术来分析HIV-1感染的主机外来体蛋白质组的作用的定量蛋白质组学方法。该协议可以很容易地适应于不同的压力或感染的条件下的细胞。
Abstract
Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.
Introduction
许多人类疾病，包括病毒感染，通常与所发生和周围的影响的细胞独特的细胞过程有关。蛋白质，经常充当最终细胞效应，调解这些进程。蛋白质的分析往往可以提供宝贵的资料，受影响细胞的局部环境，并帮助我们理解疾病的发病机制的基本机制。在各种蛋白质分析技术，蛋白质组学拥有特别大的承诺。作为一个强大的，大规模的工具，蛋白质组学可以提供细胞过程的全身性的了解，特别是在功能和蛋白质相互作用的区域。判断特定蛋白质是通过标记技术，它允许研究者监测细胞成分，特别是蛋白质的表达的发展作出了简单，在调查的部位。虽然许多蛋白质组学分析都在细胞被执行蛋白质组的规模，对亚细胞区室蛋白质组学表征已被证明是特别信息1。这是在HIV-1感染的研究很好的例子。
外来体，通过广泛的细胞类型2，3，分泌30-100纳米膜囊泡间通讯以及分子运输的关键组成部分。他们先前发现的HIV-1出芽过程4,5发挥重要作用。通过蛋白质组学分析与解剖功能结合起来，我们发现，从HIV-1感染的细胞释放的外来体是由一个独特的和定量不同的蛋白质的签名和港口监管分子在邻国接受细胞，包括细胞凋亡与增殖6影响细胞特性的。该方法在此协议中描述，即SILAC从HIV-1感染细胞的外来体7基于蛋白质组学表征（在细胞培养物中的氨基酸稳定同位素标记）。类似的方法可以适用于通过调整试验应力的特定隔室或感兴趣的级分，使对所描述的程序进行必要的更改更好地理解发病期间其他亚细胞区室。
鉴于最近的定量蛋白质组学方法的发展，有许多从选择最有效的方法为一个特定的实验时选择。其中包括基于化学的iTRAQ（用于相对和绝对定量的等压标记）8和无标记MRM（多反应监测）9技术。这两种方法是强大的工具，并且是特定的设置很好的选择。对于一个典型的实验室主要与细胞系的工作，然而，这两种方法具有relativelÿ较高的成本，而且更费时相比SILAC基础的方法时。 SILAC是一个基于代谢标记技术并入的氨基酸从培养基进入细胞蛋白质的非放射性的同位素形式。通常情况下，SILAC实验开始与两种细胞群体，例如，感染和未感染的。每个不同标记在其特定的同位素环境满为止标注的实现。然后这些细胞的标记的外来体经受蛋白提取。一旦萃取，将标记的外来体的蛋白质是使用液相色谱串联质谱法10进行分析。最后，质谱结果和显著标记的蛋白质经受统计和生物信息学分析以及严格生物化学验证。我们以前的研究报告表明，SILAC /外来体程序更适合的细胞系比初级细胞，如细胞系通常是在一个活跃的增殖状态高效同位素标记，
Protocol
Representative Results
Discussion
在本文中描述的方法，我们证明了SILAC技术的应用来调查HIV-1感染的主机上的外来体蛋白质组的效果。最初，未感染和HIV-1感染的细胞中差异同位素标记。然后差异标记外泌体进行蛋白质提取前进行纯化。接着，液相色谱 – 串联质谱法被用来分析外来体的蛋白质组。最后，将得到的质谱数据以及潜在的候选蛋白质进行统计和生物信息学的前下游生化解剖分析。
整个协议的?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由ARRA补充，寿命/塔夫茨/布朗恒虚警，P30AI042853-13S1，NIH P20GM103421，P01AA019072，R01HD072693和K24HD080539为BR支持。这项工作也是由寿命试验研究基金（＃701- 5857），罗得岛基金会医学研究资助（＃20133969），和NIH COBRE URI / RIH试点研究基金（P20GM104317）到ML支持。我们感谢詹姆斯的美荷和Vy党与手稿图制作提供帮助。
Materials
| H9 cell line | ATCC | HTB-176 | |
| Trypan Blue | Thermo Fisher | 15250061 | |
| MTT assay kit | Thermo Fisher | V13154 | |
| Dialyzed fetal bovine serum (FBS) | Thermo Fisher | 26400044 | |
| SILAC Protein Quantitation Kit – RPMI 1640 | Thermo Fisher | 89982 | DMEM version (89983) |
| L-Arginine-HCl, 13C6, 15N4 for SILAC | Thermo Fisher | 88434 | |
| L-Lysine-2HCl, 13C6 for SILAC | Thermo Fisher | 88431 | |
| HIV-1NL4-3 | NIH AIDS Reagent Program | 2480 | |
| Alliance HIV-1 p24 Antigen ELISA kit | PerkinElmer | NEK050001KT | |
| Refrigerated super-speed centrifuge | Eppendorf | 22628045 | |
| Refrigerated ultracentrifuge | Beckman Coulter | 363118 | Should be able to reach 100,000g |
| 50mL Conical Centrifuge Tubes | Thermo Fisher | 14-432-22 | |
| Ultracentrifuge Tubes | Beckman Coulter | 326823 | |
| SW 32 Ti Rotor | Beckman Coulter | 369694 | |
| RIPA buffer | Thermo Fisher | 89900 | |
| Protease Inhibitor Cocktails | Thermo Fisher | 78430 | |
| ThermoMixer | Eppendorf | 5384000020 | |
| BCA Protein Assay Kit | Thermo Fisher | 23250 | |
| Spectrophotometer | Biorad | 1702525 | |
| SDS PAGE Gel apparatus | Thermo Fisher | EI0001 | |
| Novex 4-20% Tris-Glycine Mini Gels | Novex | XV04200PK20 | |
| Gel staining reagent | Sigma Aldrich | G1041 | |
| Sequencing Grade Modified Trypsin | Promega | V5111 | |
| SpeedVac Concentrator | Thermo Fisher | SPD131DDA | |
| Antibody to human annexin A5 | Abcam | ab14196 | |
| Antibody to human lactate dehydrogenase B chain | Abcam | ab53292 | |
| Graphing and Statistical Software | Systat | SigmaPlot | Or GraphPad Prism |
| Quantitative proteomics software suite | Max Planck Institue of Biochemistry | Maxquant | |
| Software and databases | Various vendors | Refer to main text for details |
References
- Kowal, J., et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A. 113 (8), 968-977 (2016).
- Schorey, J. S., Bhatnagar, S. Exosome function: from tumor immunology to pathogen biology. Traffic. 9 (6), 871-881 (2008).
- Thery, C., Ostrowski, M., Segura, E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 9 (8), 581-593 (2009).
- Booth, A. M., et al. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol. 172 (6), 923-935 (2006).
- Van Engelenburg, S. B., et al. Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits. Science. 343 (6171), 653-656 (2014).
- Li, M., et al. Quantitative proteomic analysis of exosomes from HIV-1-infected lymphocytic cells. Proteomics. 12 (13), 2203-2211 (2012).
- Ong, S. E., et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 1 (5), 376-386 (2002).
- Ross, P. L., et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 3 (12), 1154-1169 (2004).
- Anderson, L., Hunter, C. L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol Cell Proteomics. 5 (4), 573-588 (2006).
- Ong, S. E., Mann, M. Stable isotope labeling by amino acids in cell culture for quantitative proteomics. Methods Mol Biol. 359, 37-52 (2007).
- Strober, W. . Current Protocols in Immunology. , (2001).
- Verma, A., et al. Evaluation of the MTT lymphocyte proliferation assay for the diagnosis of neurocysticercosis. J Microbiol Meth. 81 (2), 175-178 (2010).
- Cepko, C., Pear, W. Retrovirus infection of cells in vitro and in vivo. Curr Protoc Mol Biol. , (2001).
- Lennette, E. T., Karpatkin, S., Levy, J. A. Indirect immunofluorescence assay for antibodies to human immunodeficiency virus. J Clin Microbiol. 25 (2), 199-202 (1987).
- Thery, C., Amigorena, S., Raposo, G., Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. , 22 (2006).
- Olson, B. J. S. C., Markwell, J. . Current Protocols in Protein Science. , (2001).
- Gauci, V. J., Padula, M. P., Coorssen, J. R. Coomassie blue staining for high sensitivity gel-based proteomics. J Proteom. 90, 96-106 (2013).
- Soldi, M., Bonaldi, T. The ChroP approach combines ChIP and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin. J Vis Exp. (86), (2014).
- Trompelt, K., Steinbeck, J., Terashima, M., Hippler, M. A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry. J Vis Exp. (85), (2014).
- Li, M., et al. Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication. J Clin Invest. 126 (8), 3117-3129 (2016).
- Li, M., Ramratnam, B. Proteomic Characterization of Exosomes from HIV-1-Infected Cells. Methods Mol Biol. 1354, 311-326 (2016).
- Emmott, E., Goodfellow, I. Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics. J Vis Exp. (89), (2014).
- Keerthikumar, S., et al. ExoCarta: A Web-Based Compendium of Exosomal Cargo. J Mol Biol. 428 (4), 688-692 (2016).
- Kim, D. K., et al. EVpedia: a community web portal for extracellular vesicles research. Bioinformatics. 31 (6), 933-939 (2015).
- Fu, W., et al. Human immunodeficiency virus type 1, human protein interaction database at NCBI. Nucleic Acids Res. 37, 417-422 (2009).
- Pathan, M., et al. FunRich: An open access standalone functional enrichment and interaction network analysis tool. Proteomics. 15 (15), 2597-2601 (2015).
- Huang da, W., Sherman, B. T., Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 4 (1), 44-57 (2009).
- Szklarczyk, D., et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 39, 561-568 (2011).
- Thery, C., Amigorena, S., Raposo, G., Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. , (2006).
- Lobb, R. J., et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 4, 27031 (2015).
- Webber, J., Clayton, A. How pure are your vesicles. J Extracell Vesicles. 2, (2013).
- Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol Methods. 338 (1-2), 21-30 (2008).
- Chertova, E., et al. Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol. 80 (18), 9039-9052 (2006).
- Nikolov, M., Schmidt, C., Urlaub, H. Quantitative mass spectrometry-based proteomics: an overview. Methods Mol Biol. 893, 85-100 (2012).
