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。

1.细胞培养和HIV-1感染注:在开始实验之前，建议通过MTT法12以检查细胞的通过台盼蓝染色11和生存能力它们的增殖。它也使用新制备SILAC介质的关键。各种细胞系都可以使用，只要它们是在一个主动增殖阶段，很容易受到HIV-1感染，或所选择的测试条件。在这个协议中，使用的H9细胞系作为例子。 <ol><li>种子2×10 6个 H9细胞到每个细胞培养瓶。生长一组在标有含有10％透析的胎牛血清（FBS），100毫克/升13 C 6 -L-赖氨酸和100毫克/升13 C- 6 15 N 4 L-精氨酸的RPMI 1640培养基中10毫升生长另一组在10毫升的未标记的RPMI 1640培养基中，用透析的FBS 10％，100毫克/升的L-赖氨酸和100mg / L的L-精氨酸。 </li><li>生长的细胞与重氨基酸在该点在标记的培养基中的细胞的蛋白质实际上完全标记6倍增（&gt; 99％）。 （取决于细胞类型每1-3天。对于H9细胞，改变每3天培养基）添加新鲜的培养基或定期更换介质。在标记结束时，增加培养物体积（ 例如约30毫升），以容纳更多的细胞的生长。 注:确定确切细胞在通过台盼蓝染色和细胞计数实验开始倍增时间。 </li><li>感染标记细胞HIV-1 NL4-3使用标准的HIV-1感染的协议13 48小时用。孵育适当量的病毒与感染的周围0.3复数（MOI）将细胞。通过使用HIV-1 p24抗原ELISA试剂盒培养上清液的P24定量检查HIV-1感染。执行免疫荧光法14来确认细胞的成功感染。记p在不断增加中未标记的未标记的细胞无HIV-1感染。 </li><li>在感染结束时（步骤2.1）收获两组上清液。 </li></ol> 2.外来体隔离注:通过一系列的步骤，超速离心从培养上清液外体组分富集15。执行在4℃以下所有步骤，用超速离心机转子，可以达到至少100,000×g的速度。 <ol><li>收集培养物上清液在50ml锥形管中，避免了细胞。离心他们在300 xg离心10分钟以除去剩余的细胞。 </li><li>收集在新50ml锥形管中的上清液并在2000 xg离心离心它们10分钟，以去除死细胞。转让所得上清液商业转子兼容管是能够承受超离心。 </li><li>一定要平衡超速离心管。离心30分钟试管以10,000 xg离心以除去细胞碎片。 </li><li>收集70分钟的超速离心管上清和离心机在100,000×g的。弃去上清液。 </li><li>悬浮丰富的外来体颗粒在5毫升新鲜的PBS。在100,000×g的重新传送解决方案，以新鲜的超速离心管，离心70分钟。 </li><li>丢弃上清液。来存储外来体长期，悬浮颗粒在50μlPBS中并存储在-80℃。或者，如下所示直接进行蛋白质提取。 </li></ol> 3.蛋白提取和准备<ol><li>溶解在添加了蛋白酶抑制剂鸡尾酒100-200微升的RIPA裂解和提取缓冲液分离外来体粒料。缓冲区是由25毫摩尔Tris-盐酸盐，150mM氯化钠，1％NP-40，1％脱氧胆酸钠和0.1％SDS（十二烷基硫酸钠）的，在pH 7.6。蛋白酶抑制剂鸡尾酒应含有多种蛋白酶抑制剂，如抑肽酶，苯丁抑制素，升eupeptin，胃蛋白酶抑制素A和EDTA（乙二胺四乙酸），能够抑制全方位的蛋白酶。 </li><li>以13,000 xg离心（4℃）离心10分钟溶解的解决方案，以及清除上清液转移至新的1.5 ml微量离心管。 </li><li>量化使用二辛可宁酸（BCA）或Bradford测定16的外来体的各样品的蛋白质浓度。 </li><li>混合蛋白质从标记和未标记的样品等量（2微克），并在120毫安/运行在4-20％SDS-PAGE凝胶的等量混合物200伏30分钟。 </li><li>随后脱色17用考马斯蓝染色凝胶。 </li><li>用刀片从凝胶上切样品车道。然后切泳道成10-15等份。把每一块到一个新的1.5 ml离心管，总共10-15管。 </li><li>沉浸在立方体25毫米NH 4 HCO 3在50％乙腈，旋涡，并丢弃上清液。重复两次。干立方体在真空浓缩器18，19。 </li><li>再水合用10mM二硫苏糖醇（DTT），涡旋，并短暂离心立方体。孵育在56℃进行1小时。弃上清。 </li><li>沉浸凝胶立方体55毫米碘乙酰胺，漩涡和旋转。在室温下孵育在黑暗中45分钟。弃上清。 </li><li>浸入25毫米NH 4 HCO 3，旋涡，旋转立方体，弃上清。 </li><li>沉浸在立方体25毫米NH 4 HCO 3在50％乙腈，涡旋和旋转。重复3.9和3.10。 </li><li>干燥，在真空浓缩的立方体。添加25μl的测序级改良的胰蛋白酶在25mM的NH 4 HCO 3，孵育在4℃下进行30分钟，并弃去过量的溶液。浸入25毫米NH 4 HCO 3立方体没有胰蛋白酶，和孵育过夜在37℃。 </li><li>旋转简要立方体下来，转移所产生的肽的额外克拉到新管中。在50％乙腈中添加30微升的5％的甲酸的立方体，涡旋30分钟，和自旋。结合上清液用提取物。干燥用真空浓缩所述肽萃取到小于5微升。 </li><li>提交样品质谱核心设施用于LC-MS / MS分析。该MS分析数据，其可以从开放源码软件来产生，典型地包括用于标识的每个蛋白的登录号，标记/未标记的比率，并确定了独特肽的数量。 </li></ol> 4.免疫印迹验证注:Western印迹，建议以验证质谱结果。 <ol><li>遵循标准Western印迹协议和保证蛋白的从各组等量装载。使用由MS来识别针对目标蛋白的抗体（ 例如膜联蛋白A5，乳酸脱氢酶B链）。免疫印迹检测，符合密度analysi一起S，可以重申，MS蛋白质鉴定和定量是正确的。 </li></ol> 5.蛋白质组数据分析注:每个MS复制显著蛋白质候选人的数据质量评估，数据预处理，计算和确定单独完成。一旦上述分析完成后，从重复分析的数据进行比较和组合6，20，21。 <ol><li>评估MS数据的质量。 <ol><li>首先，LOG2改造SILAC-MS标记/定量蛋白质未标记的比例在电子表格程序。 </li><li>在科学绘图和统计软件，组的比例为40-100比箱并绘制每仓比的数目，以产生一个直方图。直方图的正态分布表示MS数据6的优良品质。做到这一步对所有MS复制。 </li></ol></li><li>为了提高MS肽比率的信心和准确性，考虑取消有不到两个量化肽蛋白。 </li><li>要确定显著上调和下调的蛋白质的考生，请使用以下步骤计算阈值的意义22。 <ol><li>在科学绘图和统计软件，生成一个非线性回归或曲线拟合到频率分布数据。此步骤产生可用于计算截止值的值。计算的截止值作为平均±1.96σ为95％置信限或2.56σ为99％置信限。 注:计算临界值的具体情况可以在艾默特等人发现。 22。 </li><li>选择的候选蛋白质的比例，其要么是大于1.96（或2.56）的标准偏差的中位数（显著过表达）高于或低于1.96（或2.56）标准差低于中位数（显著下表达）。 </li></ol></li><li>比较上述标题显著候选人的重复来实现的一致性。确保它们符合以下条件，通过这最后的选择步骤:考生必须在所有重复被一致确定;和候选的重复必须在其调节的方向是一致的（优选地，至少2选自候选的3个重复的要么是上调或下调）。 </li><li>最终确定由合并他们重复&#39;数据符合上述标准的候选人的数据。 </li></ol> 6.生物信息学检验和检定注:现有基因组和生物信息学的信息提供了丰富的信息，几乎所有的蛋白质。数据挖掘和生物信息学分析这些信息可以深入了解大量进入显著候选人的财产和功能的帮助。这PROCESS通常是必要的，以设计恰当下游湿实验室实验。 <ol><li>使用他们的GenBank登记号或的UniProt的ID，搜索针对当前外来体数据库的候选人（Exocarta 23 Evpedia 24），以验证该候选蛋白已在外来体先前已经找到。这一步增加了信心层的考生确实在外来体。 <ol><li>访问http://www.exocarta.org/，点击"查询"输入无论是基因或蛋白质名称/登录号。摘要页面将出现，并在外来体的证据将显示，提供，如果有任何。 </li></ol></li><li>搜索针对HIV-1和人类蛋白质相互作用数据库25的候选者。可替代地，搜索特定数据库能够提供关于所述候选蛋白质的相互作用和测试条件的信息。 <ol><li>在www.ncbi.nlm.nih.gov/RefSeq/HIV访问数据库互动。在"蛋白质域名"条目，输入候选人的名称或登录号，然后点击搜索。搜索结果可以提供深入的HIV-1与蛋白质候选人之间的相互作用，并提出它的候选人可能是真正的HIV-1相关联。 </li></ol></li><li>进口GenBank登记号或考生进入GO分析软件（如FunRich，功能丰富的分析工具26）的UniProt ID和运行该软件。 <ol><li>下载在http://www.funrich.org/软件。安装完成后，打开软件，在富集分析，单击"添加数据集"，并上传的蛋白质/基因名单。接下来，选择图表类型的可视化分析GO。 注:GO的分析结果将在饼图的形式显示。此步骤有助于我们获得与生物过程（BP），蜂窝组件等领域的考生有关环球透视（CC）和分子功能（MF）。 </li></ol></li><li>访问DAVID在http://david.ncifcrf.gov/ 27，点击其网站上的"功能性注释工具"。进入候选列表中，选择基因"标识"，如"的UniProt ID"，选择"基因名单"和搜索。富集的GO术语，p值等参数可以通过点击在"Gene_Ontology"类别中的"注释摘要结果"页面中找到。 </li><li>调查与其他蛋白质的候选潜在的相互作用，可以使用公开地可访问STRING数据库28阐明潜在的蛋白-蛋白相互作用和可能的生化途径。 注:GO分析和功能注释（6.3-6.4节），也可以使用最新的软件STRING执行。 <ol><li>在http://string-db.org/访问数据库。输入的蛋白质ID或子序列插入为S操作搜索框，然后选择分析正确的物种。点击"搜索"。结果会就直接（物理）和间接（功能）协会的信息。对于外来体的候选人的十大知名匹配结果将显示，并应考虑显著候选人的选择。 注:与考生相关的大量信息可以使用上面的步骤进行分析。最显著候选可以被选择用于进一步下游分子和生化分析。 </li></ol></li></ol>

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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=EVpedia:+a+community+web+portal+for+extracellular+vesicles+research.">EVpedia: a community web portal for extracellular vesicles research.</a> Bioinformatics. 31 (6), 933-939 (2015).</li><li>Fu, W., 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=Human+immunodeficiency+virus+type+1,+human+protein+interaction+database+at+NCBI.">Human immunodeficiency virus type 1, human protein interaction database at NCBI.</a> Nucleic Acids Res. 37, 417-422 (2009).</li><li>Pathan, M., 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=FunRich:+An+open+access+standalone+functional+enrichment+and+interaction+network+analysis+tool.">FunRich: An open access standalone functional enrichment and interaction network analysis tool.</a> Proteomics. 15 (15), 2597-2601 (2015).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat Protoc. 4 (1), 44-57 (2009).</li><li>Szklarczyk, D., 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=The+STRING+database+in+2011:+functional+interaction+networks+of+proteins,+globally+integrated+and+scored.">The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.</a> Nucleic Acids Res. 39, 561-568 (2011).</li><li>Thery, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , (2006).</li><li>Lobb, R. J., 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=Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma.">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> J Extracell Vesicles. 4, 27031 (2015).</li><li>Webber, J., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+pure+are+your+vesicles.">How pure are your vesicles.</a> J Extracell Vesicles. 2, (2013).</li><li>Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+between+exosomes+and+HIV-1:+purification+of+both+vesicles+from+cell-free+supernatants.">Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants.</a> J Immunol Methods. 338 (1-2), 21-30 (2008).</li><li>Chertova, 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=Proteomic+and+biochemical+analysis+of+purified+human+immunodeficiency+virus+type+1+produced+from+infected+monocyte-derived+macrophages.">Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.</a> J Virol. 80 (18), 9039-9052 (2006).</li><li>Nikolov, M., Schmidt, C., Urlaub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry-based+proteomics:+an+overview.">Quantitative mass spectrometry-based proteomics: an overview.</a> Methods Mol Biol. 893, 85-100 (2012).</li></ol>

The authors have declared no conflict of interest.

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?...

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，

<table><tbody><tr><td>H9 cell line</td><td>ATCC</td><td>HTB-176</td><td></td></tr><tr><td>Trypan Blue</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>MTT assay kit</td><td>Thermo Fisher</td><td>V13154</td><td></td></tr><tr><td>Dialyzed fetal bovine serum (FBS)</td><td>Thermo Fisher</td><td>26400044</td><td></td></tr><tr><td>SILAC Protein Quantitation Kit &amp;ndash; RPMI 1640</td><td>Thermo Fisher</td><td>89982</td><td>DMEM version (89983)</td></tr><tr><td>L-Arginine-HCl, &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;, &lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;4&lt;/sub&gt; for SILAC</td><td>Thermo Fisher</td><td>88434</td><td></td></tr><tr><td>L-Lysine-2HCl, &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt; for SILAC</td><td>Thermo Fisher</td><td>88431</td><td></td></tr><tr><td>HIV-1 NL4-3</td><td>NIH AIDS Reagent Program</td><td>2480</td><td></td></tr><tr><td>Alliance HIV-1 p24 Antigen ELISA kit</td><td>PerkinElmer</td><td>NEK050001KT</td><td></td></tr><tr><td>Refrigerated super-speed centrifuge</td><td>Eppendorf</td><td>22628045</td><td></td></tr><tr><td>Refrigerated ultracentrifuge</td><td>Beckman Coulter</td><td>363118</td><td>Should be able to reach 100,000 x g</td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>Thermo Fisher</td><td>14-432-22</td><td></td></tr><tr><td>Ultracentrifuge Tubes</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>SW 32 Ti Rotor</td><td>Beckman Coulter</td><td>369694</td><td></td></tr><tr><td>RIPA buffer</td><td>Thermo Fisher</td><td>89900</td><td></td></tr><tr><td>Protease Inhibitor Cocktails</td><td>Thermo Fisher</td><td>&amp;nbsp;78430</td><td></td></tr><tr><td>ThermoMixer&amp;nbsp;</td><td>Eppendorf</td><td>5384000020</td><td></td></tr><tr><td>BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Fisher</td><td>23250</td><td></td></tr><tr><td>Spectrophotometer</td><td>Biorad</td><td>1702525</td><td></td></tr><tr><td>SDS PAGE Gel apparatus</td><td>Thermo Fisher</td><td>EI0001</td><td></td></tr><tr><td>Novex 4-20% Tris-Glycine Mini Gels</td><td>Novex</td><td>XV04200PK20</td><td></td></tr><tr><td>Gel staining reagent</td><td>Sigma Aldrich</td><td>G1041</td><td></td></tr><tr><td>Sequencing Grade Modified Trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>SpeedVac Concentrator</td><td>Thermo Fisher</td><td>SPD131DDA</td><td></td></tr><tr><td>Antibody to human annexin A5</td><td>Abcam</td><td>ab14196</td><td></td></tr><tr><td>Antibody to human lactate dehydrogenase B chain</td><td>Abcam</td><td>ab53292</td><td></td></tr><tr><td>Graphing and Statistical Software</td><td>Systat&amp;nbsp;</td><td>SigmaPlot&amp;nbsp;</td><td>Or GraphPad Prism</td></tr><tr><td>Quantitative proteomics software suite</td><td>Max Planck Institue of Biochemistry</td><td>Maxquant&amp;nbsp;</td><td></td></tr><tr><td>Software and databases</td><td>Various vendors</td><td></td><td>Refer to main text for details</td></tr></tbody></table>

silac based proteomic characterization of exosomes from hiv-1 infected cells

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.

图1A是概述SILAC标记程序21的流程图。为了净化外来体，样品必须通过离心纺丝下来。 图1B示出了通过串行超速离心21外来体纯化的步骤。一旦纯化，如在程序中概述的外来体受实验蛋白质组学分析。 图2A是用于从蛋白质组数据21确定显...

Watch this Scientific Journal Video about SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells at JoVE.com

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

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。

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

silac-based-proteomic-characterization-exosomes-from-hiv-1-infected

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.6K 次观看.  Brown University. 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。 

视频: SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

科研

生物化学

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

免疫与感染

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

生物学

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

猕猴免疫缺陷病毒感染 CD4+ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

使用多参数光伏平台对钙流入和HIV-1感染进行单细胞特征描述

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

的蛋白质相互作用的合作伙伴在哺乳动物细胞中使用SILAC-免疫定量蛋白质组学鉴定

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...