Name: lipid droplet isolation for quantitative mass spectrometry analysis
Uploaded: 2017-04-17T13:00:00.000+00:00
Duration: 10 min 23 s
Description: Watch this Scientific Journal Video about Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis at JoVE.com

我们感谢R. Bartenschlager（海德堡大学）的JC1结构，CM赖斯（洛克菲勒大学）的Huh7.5细胞，J·麦克劳奇伦（医学研究理事会病毒学单元）为JFH1建造，T·韦克塔（国家研究所传染病，日本）的JFH1，和B·韦伯斯特和克·格林（病毒学研究所格莱斯顿和免疫学）为HCVcc的记者结构。这项工作是由从DFG资金支持（HE 6889 / 2-1（EH），INST 337 / 15-1 2013，和INST 337 / 16-1 2013（HS））。海因里希Pette研究所，莱布尼兹研究所实验病毒学由自由汉萨城汉堡和健康的联邦支持。该资助者在研究设计，数据收集和分析，决定发表或准备手稿没有作用。

1.介质的制备稳定同位素标记与氨基酸在细胞培养（SILAC） 注意:在此，SILAC蛋白定量试剂盒- DMEM补充有50毫克13 C 6 L-精氨酸盐酸用于SILAC标记。经透析的胎牛血清（FCS）具备SILAC蛋白定量试剂盒。 <ol><li>从DMEM培养基的每个瓶子中取出50毫升，并加入50毫升透析FCS的。 </li><li>溶解50毫克13 C 6 L-赖氨酸2HCl的和在1毫升培养基的50毫克13 C 6 L-精氨酸HCl中。调匀氨基酸添加到DMEM + FCS中。 </li><li>添加1X青霉素/链霉素和1x L-谷氨酰胺的替代品。无菌过滤器使用0.45μm的过滤器介质。标签瓶"重" SILAC中。 </li><li>使用50毫克L-精氨酸盐酸和50mg L-赖氨酸2HCl的1.3 - 以制备"光"介质，重复步骤1.1。标签的瓶子"光" SILAC平台。 </li></ol> 2. SILAC标签和氨基酸掺入控制<ol><li>胰蛋白酶化的细胞（1×胰蛋白酶-EDTA）和分割1×10 5个的Huh7.5细胞分成2个孔含有2毫升培养基中，一个孔与"重" SILAC介质和一个与"光"的6孔培养板的SILAC培养基。 </li><li>培养物中的细胞至少6代（分流比:1:6）;后6代，"重"氨基酸的掺入应超过95％。 </li><li>收获"重"的1×10 6个细胞-和"光" -标记的细胞群来分析掺入效力。洗在1×PBS中的细胞，并通过离心在160×g离心5分钟，4℃沉淀细胞。 </li><li>重悬细胞沉淀中的MS-缓冲液（150mM的NaCl，50mM的Tris-盐酸pH为7.4，和1mM EDTA补充有1X蛋白酶抑制剂混合物）150μL，并在冰上孵育细胞30分钟。裂解的C厄尔通过超声处理，在4℃。 注:以下是本文所用的超声设置:定时器，持有;输出控制，8;占空比，80％;和2×30个脉冲。 </li><li>在11000×g下4℃下除去通过离心细胞碎片10分钟。将上清转移到新管中，并确定与洗涤剂相容的蛋白质测定蛋白质浓度。 </li><li>混合75微克蛋白的用6×样品缓冲液（375毫摩尔的Tris-HCl，pH为6.8，25.8％甘油，123毫克/毫升SDS，600微克/毫升溴酚蓝，和60μL/ mL的β巯基乙醇），煮沸，在95℃下进行5分钟，和运行缓冲液（3.02克/ L Tris碱18.8克/升甘氨酸，和1g / L SDS）在180 V的大约1个小时或根据制造商的通过SDS-PAGE的蛋白质分离在SDS "指令。 </li><li>转移到凝胶胶体考马斯染色溶液。从每个车道消费相同的蛋白质带。消化的蛋白质用胰蛋白酶和分析由MS analysi掺入效力S，如所描述的22。 </li></ol> 3. SILAC标记的Huh7.5细胞的脂滴分离<ol><li>感染细胞的一个群体（ 即那些与"光"氨基酸标示）通过与病毒原温育（ 例如，JC1 NS5AB-mKO2-BSD，MOI 1）在37℃下4小时，如所描述的21与HCV报告病毒。 注:JC1 NS5AB-mKO2-BSD是HCV病毒携带荧光记者（单体Kusabira橙2，mKO2）监测感染率接着重复的NS5A-NS5B裂解位点之间的灭瘟抗性基因（BSD），先前所描述的21， 23。与HCV工作需要BSL2 +（美国）或S3 **（德国）生物安全级别的遏制和做法。相反，感染HCV的细胞可以感染不同的病原体。 </li><li>展开HCV感染的"轻"细胞与未感染的＆＃34;重"细胞在培养物中的传代，固定用PBS中的4％多聚甲醛（PFA）的等分试样，并通过荧光标记蛋白的流式细胞术（ 例如，mKO2），确定如所描述的21中的HCV感染率;所述感染率应该是脂滴隔离注高于90％:如果使用JC1 NS5AB-mKO2-BSD HCV株，10微克/毫升杀稻瘟菌素S添加到"光"介质选择为HCV阳性细胞。 </li><li> 1天前的脂滴隔离，洗涤细胞，用PBS，胰蛋白酶化，再悬浮于"轻"和"重"介质，和计数使用Neubauer计数室中的细胞。种子在150 cm 2的细胞培养皿各人口的7×10 6细胞。每次准备"轻"和"重"的细胞群至少5种菜肴。培养细胞在30mL的介质/皿O / N。 </li><li>除去培养基并洗涤细胞在1X PBS中。在拆下1X细胞PBS使用细胞刮。 </li><li>计数使用Neubauer计数室两种细胞群，并在50mL离心管池相等的细胞数目。沉淀细胞通过离心在160×g离心5分钟，4℃。 </li><li>除去PBS和重悬细胞沉淀在1ml蔗糖缓冲液（0.25M蔗糖，1mM EDTA中，和1mM DTT，补充有蛋白酶抑制剂混合物）的。细胞悬液转移到紧身Dounce匀浆并在冰上200个笔划裂解细胞。 注意:使用台盼蓝染色确认完成细胞裂解。 </li><li>转移裂解液到1.5mL微量离心管中，并在1,000×g下4℃降速细胞核和细胞碎片10分钟。 </li><li>离心后，在-20℃下作为输入控制存储后细胞核部分的25μL（PNS）的等分试样。 </li><li>放置PNS馏分的其余部分在离心管（11×60毫米）的下部，并用脂滴洗涤缓冲液（覆盖约3mL; 4mL的托特人）（50mM的磷酸钾缓冲液pH 7.4，100mM的氯化钾，1mM的EDTA，和1mM苯甲基磺酰氟）。 </li><li>离心机以100,000×g 2小时，4℃。 （ - 500μL，取决于脂滴的量大约250）从所述管的顶部收获使用弯曲，钝头插管浮动脂滴级分。放置脂滴级分在离心管（11×60毫米），覆盖与脂滴洗涤缓冲液（〜3.5毫升; 4毫升总），并重复离心步骤。 </li><li>从管的顶部收获使用弯曲，钝头插管浮动脂滴级分和脂滴转移至新的1.5mL微量离心管中。添加脂滴的洗涤缓冲液和自旋的500μL20分钟在21000×g下4℃。 注:脂滴出现，为白色带浮在缓冲的顶部。 </li><li>使用凝胶装载移液管尖端取下下卧的洗涤缓冲液，并重复此洗涤步骤三次。 </li><li>最后去除使用凝胶装载枪头洗涤缓冲液后，混合5脂滴级分μL用NP-40裂解缓冲液的10μL（50mM的Tris-盐酸pH值7.4，150mM的NaCl和1％NP-40 ，补充有蛋白酶抑制剂混合物）。在冰上孵育样品1小时，如果与传染性物质合作，以灭活该病毒。确定与洗涤剂相容的蛋白质测定蛋白质水平;蛋白的至少35微克需要用于MS分析。 </li><li>存储在-20℃的脂滴级分。 </li><li>混合具有4倍样染料脂滴级分的相应量。在冰上孵育1个小时，如果有传染性物质的工作，以使病毒失活。沸腾在95℃下5分钟。 </li><li>分离根据制造商的方案，使用SDS-PAGE的蛋白质。转移到凝胶胶体考马斯染色溶液。消费税从凝胶中的所有蛋白条带。胰蛋白酶凝胶内消化24 &lt;后/ SUP&gt;，蒸发样品和在0.1％甲酸溶解。通过LC-MS / MS分析。 注意:在此，LC-MS / MS分析在四极杆 - 时间 - 飞行质谱仪（Q-TOF）或上的线性阱四极杆（LTQ）轨道阱质谱仪上进行。这两种仪器均加上一个ESI-源到纳米UPLC系统。数据分析和LC-MS / MS在Q-TOF和上轨道阱质谱仪分析如所描述的21，22进行。请特别注意不要污染与人角蛋白样品。始终使用无角蛋白的枪头和管子。准备了免费角蛋白化学品的层流罩下的缓冲区。使用前，请用蒸馏水和乙醇所有表面和设备。一定要穿戴手套和白大褂。 </li></ol> 4.脂滴纯度分析<ol><li>稀脂滴级分1的等分试样:2和输入级分（参见步骤3.9）1:10用NP-40赖氨酸为缓冲区。在冰上孵育样品1个小时，如果有传染性物质的工作，以使病毒失活。确定与洗涤剂相容的蛋白质测定蛋白质水平。 </li><li>混合用6×样品缓冲液蛋白的等量和孵育样品在冰上1个小时如果与传染性物质合作，以灭活该病毒。用SDS-PAGE进行，并且在80伏由罐印迹蛋白质转移到硝酸纤维素膜上90分钟。 注:在封闭缓冲液（在TBS-T（10mM的Tris-盐酸pH值7.6，150mM的NaCl和0.05％吐温20）的5％脱脂干奶粉）阻挡膜后，孵育用针对脂滴标记物的抗体（ 例如，PLIN1，PLIN2，或PLIN3）和其他亚细胞区室（的标记物例如，CALR，MnSOD的，或微管蛋白），随后二次HRP偶联的抗体。使用化学发光检测蛋白质。 </li></ol>

<ol>
	<li>Thiam, A. R., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biophysics+and+cell+biology+of+lipid+droplets.">The biophysics and cell biology of lipid droplets.</a> Nat Rev Mol Cell Biol. 14 (12), 775-786 (2013).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+roles+for+lipid+droplets.">Expanding roles for lipid droplets.</a> Curr Biol. 25 (11), R470-R481 (2015).</li><li>Brasaemle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+adipocyte+biology.+The+perilipin+family+of+structural+lipid+droplet+proteins:+stabilization+of+lipid+droplets+and+control+of+lipolysis.">Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis.</a> J Lipid Res. 48 (12), 2547-2559 (2007).</li><li>Choudhary, V., Ojha, N., Golden, A., Prinz, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+family+of+proteins+facilitates+nascent+lipid+droplet+budding+from+the+ER.">A conserved family of proteins facilitates nascent lipid droplet budding from the ER.</a> J Cell Biol. 211 (2), 261-271 (2015).</li><li>Kory, N., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Fat:+Mechanisms+of+Protein+Localization+to+Lipid+Droplets.">Targeting Fat: Mechanisms of Protein Localization to Lipid Droplets.</a> Trends Cell Biol. 26 (7), 535-546 (2016).</li><li>Hashemi, H. F., Goodman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+lipid+droplets.">The life cycle of lipid droplets.</a> Curr Opin Cell Biol. 33, 119-124 (2015).</li><li>Gao, Q., Goodman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipid+droplet-a+well-connected+organelle.">The lipid droplet-a well-connected organelle.</a> Front Cell Dev Biol. 3, 49 (2015).</li><li>Herker, E., Ott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+lipid+droplets+in+host/pathogen+interactions.">Emerging role of lipid droplets in host/pathogen interactions.</a> J Biol Chem. 287 (4), 2280-2287 (2012).</li><li>Wedemeyer, H., Dore, G. J., Ward, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+on+HCV+disease+burden+worldwide+-+filling+the+gaps.">Estimates on HCV disease burden worldwide - filling the gaps.</a> J Viral Hepat. 22 Suppl 1, 1-5 (2015).</li><li>Paul, D., Madan, V., Bartenschlager, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+RNA+replication+and+assembly:+living+on+the+fat+of+the+land.">Hepatitis C virus RNA replication and assembly: living on the fat of the land.</a> Cell Host Microbe. 16 (5), 569-579 (2014).</li><li>Lindenbach, B. D., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+hepatitis+C+virus+entry+and+assembly.">The ins and outs of hepatitis C virus entry and assembly.</a> Nat Rev Microbiol. 11 (10), 688-700 (2013).</li><li>Barba, G., 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=Hepatitis+C+virus+core+protein+shows+a+cytoplasmic+localization+and+associates+to+cellular+lipid+storage+droplets.">Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets.</a> Proc Natl Acad Sci U S A. 94 (4), 1200-1205 (1997).</li><li>Shi, S. T., 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=Hepatitis+C+virus+NS5A+colocalizes+with+the+core+protein+on+lipid+droplets+and+interacts+with+apolipoproteins.">Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins.</a> Virology. 292 (2), 198-210 (2002).</li><li>Herker, 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=Efficient+hepatitis+C+virus+particle+formation+requires+diacylglycerol+acyltransferase-1.">Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1.</a> Nat Med. 16 (11), 1295-1298 (2010).</li><li>Camus, G., 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=Diacylglycerol+acyltransferase-1+localizes+hepatitis+C+virus+NS5A+protein+to+lipid+droplets+and+enhances+NS5A+interaction+with+the+viral+capsid+core.">Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid droplets and enhances NS5A interaction with the viral capsid core.</a> J Biol Chem. 288 (14), 9915-9923 (2013).</li><li>Miyanari, Y., 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+lipid+droplet+is+an+important+organelle+for+hepatitis+C+virus+production.">The lipid droplet is an important organelle for hepatitis C virus production.</a> Nat Cell Biol. 9 (9), 1089-1097 (2007).</li><li>Boulant, S., Targett-Adams, P., McLauchlan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+the+association+of+hepatitis+C+virus+core+protein+with+lipid+droplets+correlates+with+a+loss+in+production+of+infectious+virus.">Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus.</a> J Gen Virol. 88 (Pt 8), 2204-2213 (2007).</li><li>Vogt, D. A., 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=Lipid+droplet-binding+protein+TIP47+regulates+hepatitis+C+Virus+RNA+replication+through+interaction+with+the+viral+NS5A+protein.">Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein.</a> PLoS Pathog. 9 (4), e1003302 (2013).</li><li>Ploen, 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=TIP47+plays+a+crucial+role+in+the+life+cycle+of+hepatitis+C+virus.">TIP47 plays a crucial role in the life cycle of hepatitis C virus.</a> J Hepatol. 58 (6), 1081-1088 (2013).</li><li>Ploen, 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=TIP47+is+associated+with+the+hepatitis+C+virus+and+its+interaction+with+Rab9+is+required+for+release+of+viral+particles.">TIP47 is associated with the hepatitis C virus and its interaction with Rab9 is required for release of viral particles.</a> Eur J Cell Biol. 92 (12), 374-382 (2013).</li><li>Rosch, 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=Quantitative+Lipid+Droplet+Proteome+Analysis+Identifies+Annexin+A3+as+a+Cofactor+for+HCV+Particle+Production.">Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production.</a> Cell Rep. 16 (12), 3219-3231 (2016).</li><li>Kwiatkowski, 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=Ultrafast+extraction+of+proteins+from+tissues+using+desorption+by+impulsive+vibrational+excitation.">Ultrafast extraction of proteins from tissues using desorption by impulsive vibrational excitation.</a> Angew Chem Int Ed Engl. 54 (1), 285-288 (2015).</li><li>Webster, B., Ott, M., Greene, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evasion+of+superinfection+exclusion+and+elimination+of+primary+viral+RNA+by+an+adapted+strain+of+hepatitis+C+virus.">Evasion of superinfection exclusion and elimination of primary viral RNA by an adapted strain of hepatitis C virus.</a> J Virol. 87 (24), 13354-13369 (2013).</li><li>Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes.">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nat Protoc. 1 (6), 2856-2860 (2006).</li><li>Ting, L., 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=Normalization+and+statistical+analysis+of+quantitative+proteomics+data+generated+by+metabolic+labeling.">Normalization and statistical analysis of quantitative proteomics data generated by metabolic labeling.</a> Mol Cell Proteomics. 8 (10), 2227-2242 (2009).</li><li>Brasaemle, D. L., Dolios, G., Shapiro, L., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+proteins+associated+with+lipid+droplets+of+basal+and+lipolytically+stimulated+3T3-L1+adipocytes.">Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.</a> J Biol Chem. 279 (45), 46835-46842 (2004).</li><li>Tingting, P., Caiyun, F., Zhigang, Y., Pengyuan, Y., Zhenghong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subproteomic+analysis+of+the+cellular+proteins+associated+with+the+3'+untranslated+region+of+the+hepatitis+C+virus+genome+in+human+liver+cells.">Subproteomic analysis of the cellular proteins associated with the 3' untranslated region of the hepatitis C virus genome in human liver cells.</a> Biochem Biophys Res Commun. 347 (3), 683-691 (2006).</li><li>Ariumi, Y., 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=DDX3+DEAD-box+RNA+helicase+is+required+for+hepatitis+C+virus+RNA+replication.">DDX3 DEAD-box RNA helicase is required for hepatitis C virus RNA replication.</a> J Virol. 81 (24), 13922-13926 (2007).</li><li>Weinlich, S., 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=IGF2BP1+enhances+HCV+IRES-mediated+translation+initiation+via+the+3'UTR.">IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3'UTR.</a> RNA. 15 (8), 1528-1542 (2009).</li></ol>

作者什么都没有透露。

在这里，我们描述了一种协议来隔离脂滴定量脂滴的蛋白质组分析来比较具有不同的培养条件，如病毒感染下脂滴相关的蛋白质的富集和耗尽。作为一种替代的方法，所述蛋白质组分析可以与基于总的峰强度的无标记的量化来执行。这个方法没有动态范围的限制，避免了代谢问题。的SILAC方法的优点是，将样品汇集之前脂滴隔离，因此，结果是独立的错误在样品制备，消化，和LC-MS / MS分析。我们?...

<html>脂滴高度的中性脂质的核心组成的动态细胞质（和核）细胞器（甘油三酯（TG）和胆固醇酯（CE））通过磷脂单层具有嵌入蛋白1包围。所有类型的细胞产生的脂滴，但它们在大小，脂质组成，和蛋白质装饰变化。脂滴满足不同的功能，包括作为能量和膜前体贮存器或作为蛋白质沉积物。此外，通过脂类的摄取，它们防止脂毒性细胞，释放脂质作为信号分子，并参与蛋白质的降解和内质网（ER）应激2响应。因此，蛋白质的宿主结合脂滴和管理其生成，降解，贩卖，和相互作用与其他细胞器。其中有善意的脂滴结合蛋白的围脂滴蛋白家族（PLIN1-5）F"&gt; 3。 脂滴的生物合成可能开始于ER，其中ER驻留酶催化膜双层内累积中性脂质的合成，从而形成中性脂质的透镜，这是最近在酵母4很好地可视化的方法。膜弯曲和升高的磷脂酸和二酰基甘油水平，然后认为吸引涉及磷脂生物合成的蛋白质，作为核心中性脂质和屏蔽磷脂的同时合成所需的脂质液滴产生5。驻留在ER酶窝藏跨膜结构域催化了这个过程。膨胀到大的脂滴需要不同类窝藏一个两亲性螺旋，因此可以从ER到脂滴行进脂质合成酶的活性。从脂滴脂质动员发生通过甘油三酯的局部活化e和甘油二酯的脂肪酶脂肪甘油三酯脂肪酶（ATGL）和激素敏感性脂肪酶（HSL）或由不同的自噬途径，如宏观和microlipophagy或分子伴侣介导的自噬6。脂滴与其他细胞器，诸如线粒体（用于β-氧化和脂质合成）和ER相互作用（对于脂质合成和蛋白质运输），而且还与溶酶体，核内体，并且通过细胞内细菌7诱导的空泡。事实上细菌，病毒，甚至寄生虫目标，其中8 HCV复制和持久性脂滴。 HCV感染是全世界肝病相关的发病率和死亡率的主要原因之一，占每年9约为0.5万人死亡。 HCV感染的真正数量是未知的，但最近的估计表明，130 - 150万人受到慢性感染。没有疫苗免疫Ë存在，但相比于标准的基于干扰素治疗最近批准的直接作用的抗病毒药显着提高治疗反应。然而，在世界范围内，患者的治疗很可能会因新疗法的成本非常高的限制。大约有一半的人的慢性HCV感染者发展脂肪肝（脂肪变性），其特点是脂滴在肝细胞中堆积过多的条件。有趣的是，脂滴也出现了HCV复制的重要细胞器，推定作为病毒组装站点10，11。 在HCV感染的细胞，病毒蛋白核心和NS5A定位于以依赖于甘油三酯生物合成的过程脂滴，如二酰基甘油酰基转移酶1（DGAT1）的抑制剂削弱贩卖脂滴和随后的HCV颗粒生产12 </sup&gt;，13，14，15。此外，在任一核心或NS5A的脂滴结合结构域的突变抑制HCV装配16，17上 。芯和NS5A再招所有其它病毒蛋白，以及病毒RNA复制复合物，与脂质相关的密切滴16的膜。需要成功地生产感染性病毒子代10，11的所有病毒蛋白质的一致行动。结构蛋白是病毒体的一部分，和非结构蛋白促进这个过程所需要的蛋白质 - 蛋白质相互作用。有趣的是，需要对善意脂滴结合蛋白PLIN3 / TIP47两种HCV RNA的复制和病毒颗粒18的释放，19 <suP&gt;，20。尽管有这些最新进展，该机制的细节，尤其是在HCV复制的后期病毒与宿主相互作用，依然不明确，而脂滴的确切功能未知。 在这里，我们描述了以分离的脂滴为相关蛋白的定量质谱法的方法。使用这种方法，我们发现在脂滴蛋白组HCV感染期间和深刻的变化确定膜联蛋白A3为共同分馏与脂滴，并需要有效HCV成熟21的宿主蛋白。

<table><tbody><tr><td>SILAC Protein Quantitation Kit - DMEM</td><td>Thermo Fisher</td><td>89983&amp;nbsp;</td><td></td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt; L-Arginine-HCl 50&amp;nbsp;mg</td><td>Thermo Fisher</td><td>88210</td><td></td></tr><tr><td>Roti-Load 1</td><td>Roth GmbH</td><td>K929.1</td><td></td></tr><tr><td>Roti-Blue 5x Concentrate</td><td>Roth GmbH</td><td>A152.2&amp;nbsp;</td><td></td></tr><tr><td>10x SDS-Tris-Glycine - Buffer</td><td>Geyer Th. GmbH &amp;amp; Co.KG&amp;nbsp;</td><td>A1415,0250&amp;nbsp;</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Life Technologies GmbH&amp;nbsp;</td><td>350500038&amp;nbsp;</td><td></td></tr><tr><td>Penicillin/Streptomycin Solution for Cell Culture</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>P4333-100ml&amp;nbsp;</td><td></td></tr><tr><td>DPBS 1x Dulbecco&#039;s Phosphate-buffered Saline</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>D8537&amp;nbsp;</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>T3924-100ML&amp;nbsp;</td><td></td></tr><tr><td>Sodium Chloride &lt;em&gt;BioChemica&lt;/em&gt;</td><td>AppliChem GmbH</td><td>A1149,1000</td><td></td></tr><tr><td>Tris Ultrapure&amp;nbsp;&amp;nbsp;</td><td>AppliChem GmbH</td><td>A1086,5000A&amp;nbsp;</td><td></td></tr><tr><td>EDTA &lt;em&gt;BioChemica&lt;/em&gt;</td><td>AppliChem GmbH</td><td>A1103,0250</td><td></td></tr><tr><td>Protease Inhibitor Cocktail 5 mL</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>P8340-5ML&amp;nbsp;</td><td></td></tr><tr><td>D(+)-Sucrose &lt;em&gt;BioChemica&lt;/em&gt;</td><td>AppliChem GmbH</td><td>A3935,1000</td><td></td></tr><tr><td>Hydrochloric acid (HCl) 37% pure Ph. Eur., NF</td><td>AppliChem GmbH</td><td>A0625</td><td></td></tr><tr><td>DC Protein Assay&amp;nbsp;</td><td>Bio-Rad Laboratoris GmbH&amp;nbsp;</td><td>500-0116&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>AppliChem GmbH</td><td>151339</td><td></td></tr><tr><td>SDS Ultrapure</td><td>AppliChem GmbH</td><td>A1112</td><td></td></tr><tr><td>Bromophenol blue</td><td>AppliChem GmbH</td><td>A2331</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol&amp;nbsp;</td><td>AppliChem GmbH</td><td>A4338</td><td></td></tr><tr><td>Blasticidin</td><td>Invivogen</td><td>ant-bl-1&amp;nbsp;</td><td></td></tr><tr><td>Potassium Chloride&amp;nbsp;</td><td>AppliChem GmbH</td><td>A1039</td><td></td></tr><tr><td>Phenylmethanesulfonyl Fluoride&amp;nbsp;</td><td>AppliChem GmbH</td><td>A0999</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>221309</td><td></td></tr><tr><td>Dipotassium Hydrogenphosphate</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>P3786&amp;nbsp;</td><td></td></tr><tr><td>DTT&amp;nbsp;</td><td>AppliChem GmbH</td><td>A2948</td><td></td></tr><tr><td>NP-40</td><td>AppliChem</td><td>A1694</td><td></td></tr><tr><td>TWEEN 20</td><td>AppliChem</td><td>A4974</td><td></td></tr><tr><td>Nonfat dried milk powder</td><td>AppliChem</td><td>A0830</td><td></td></tr><tr><td>Anti-ADFP/ ADRP&amp;nbsp;</td><td>abcam</td><td>ab52355</td><td></td></tr><tr><td>M6PRB1/TIP47 100 &amp;micro;g</td><td>abcam</td><td>ab47639</td><td></td></tr><tr><td>Calreticulin, pAb 200 &amp;micro;g</td><td>Enzo Life Science GmbH&amp;nbsp;</td><td>ADI-SPA-600-F&amp;nbsp;</td><td></td></tr><tr><td>Anti-&amp;beta;-Tubulin&amp;nbsp;</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>T6074 200&amp;micro;l &amp;nbsp;</td><td></td></tr><tr><td>Ethanol absolute (EtOH)</td><td>Geyer Th. GmbH &amp;amp; Co.KG&amp;nbsp;</td><td>A3678,0250 &amp;nbsp;</td><td></td></tr><tr><td>Anti-MnSOD</td><td>Enzo Life Science GmbH&amp;nbsp;</td><td>ADI-SOD-110-F</td><td></td></tr><tr><td>Anti-mouse HRP</td><td>Thermo Fisher Pierce</td><td>32430</td><td></td></tr><tr><td>Anti-rabbit HRP</td><td>Thermo Fisher&amp;nbsp; Pierce</td><td>32460</td><td></td></tr><tr><td>Amersham Hyperfilm ECL</td><td>GE Healthcare</td><td>28906836</td><td></td></tr><tr><td>Lumi-Light Western Blotting Substrate</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>12015196001</td><td></td></tr><tr><td>96-Well Cell Culture Plate</td><td>Greiner Bio-One GmbH&amp;nbsp;</td><td>655 180&amp;nbsp;</td><td></td></tr><tr><td>Terumo Syringe 1 mL</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Filtropur BT 50, 500 mL, 0.45 &amp;micro;m&amp;nbsp;</td><td>SARSTEDT&amp;nbsp;</td><td>83.1823.100&amp;nbsp;</td><td></td></tr><tr><td>Mini-PROTEAN TGX Precast Gels, Any kD resolving gel</td><td>Bio-Rad Laboratoris GmbH&amp;nbsp;</td><td>456-9034&amp;nbsp;</td><td></td></tr><tr><td>6-Well Cell Culture Plate</td><td>Greiner Bio-One GmbH&amp;nbsp;</td><td>657160&amp;nbsp;</td><td></td></tr><tr><td>Dishes Nunclon 150/20&amp;nbsp;</td><td>Fisher Scientific GmbH&amp;nbsp;</td><td>10098720 - 168381&amp;nbsp;</td><td></td></tr><tr><td>Cell Scraper</td><td>neoLab Migge GmbH&amp;nbsp;</td><td>C-8120&amp;nbsp;</td><td></td></tr><tr><td>Tube, 50 mL</td><td>Greiner Bio-One GmbH&amp;nbsp;</td><td>227261&amp;nbsp;</td><td></td></tr><tr><td>SafeSeal Tube RNase-free&amp;nbsp;</td><td>SARSTEDT&amp;nbsp;</td><td>72.706.400&amp;nbsp;</td><td></td></tr><tr><td>Ultra Clear Centrifuge Tubes 11 x 60 mm</td><td>Beckman Coulter GmbH&amp;nbsp;</td><td>344062&amp;nbsp;</td><td></td></tr><tr><td>Suction Needles</td><td>Transcodent</td><td>6482</td><td></td></tr><tr><td>Biosphere Fil. Tip 1000&amp;nbsp;</td><td>SARSTEDT&amp;nbsp;</td><td>70.762.211&amp;nbsp;</td><td></td></tr><tr><td>Biosphere Fil. Tip 200</td><td>SARSTEDT&amp;nbsp;</td><td>70.760.211&amp;nbsp;</td><td></td></tr><tr><td>Biosphere Fil. Tip 10</td><td>SARSTEDT&amp;nbsp;</td><td>70.1130.210&amp;nbsp;</td><td></td></tr><tr><td>Dounce Tissue Grinder</td><td>Fisher Scientific GmbH&amp;nbsp;</td><td>11883722</td><td></td></tr><tr><td>Pestles For Dounce All-Glass Tissue Grinders</td><td>Fisher Scientific GmbH&amp;nbsp;</td><td>10389444</td><td></td></tr><tr><td>Orbitrap Fusion</td><td></td><td></td><td></td></tr><tr><td>Branson Sonifier 450</td><td></td><td></td><td></td></tr><tr><td>Thermomixer comfort, with Thermoblock 1.5 mL</td><td>Eppendorf</td><td>5355&amp;nbsp;000.127</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module, and PowerPac Basic Power Supply,&amp;nbsp;</td><td>BioRad</td><td>165-8033</td><td></td></tr><tr><td>Mini-PROTEAN 3 Multi-Casting Chamber</td><td>BioRad</td><td>165-4110</td><td></td></tr><tr><td>PowerPac HC Power Supply</td><td>Biorad</td><td>164-5052</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Optima L-90K</td><td>Beckman Coulter GmbH&amp;nbsp;</td><td>365670</td><td></td></tr><tr><td>SW 60 Ti Rotor</td><td>Beckman Coulter GmbH&amp;nbsp;</td><td>335649</td><td></td></tr><tr><td>Infinite M1000 PRO</td><td>Tecan</td><td></td><td></td></tr></tbody></table>

lipid droplet isolation for quantitative mass spectrometry analysis

脂滴是多种不同的病原体，最突出的是丙型肝炎病毒（HCV）复制至关重要的，因为病毒粒子的形态发生的推定站点。定量脂滴的蛋白质组分析可以用于鉴定定位于或诸如病毒感染的条件下从脂滴移位蛋白质。在这里，我们描述了已经成功用于表征以下感染HCV的脂滴蛋白质组变化的协议。我们在细胞培养（SILAC）使用稳定同位素标记与氨基酸和因此标签与"重"氨基酸细胞的一个群体的完整蛋白质通过质谱定量的蛋白质。对于脂滴隔离，这两个细胞群体（ 即 HCV感染/"光"的氨基酸和未感染的对照/"重"氨基酸）的1:1混合并在低渗缓冲液进行机械裂解。除去细胞核和细胞天后通过低速离心ebris，脂滴相关蛋白通过随后在等渗缓冲液洗涤3次两个后续步骤超速离心富集。脂滴级分的纯度通过Western印迹用抗体识别不同的亚细胞区室分析。脂滴相关蛋白然后通过随后进行考马斯染色的SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离。胰蛋白酶消化后，将肽通过液相色谱 - 电喷雾离子化串联质谱（LC-ESI-MS / MS）进行定量。使用这种方法，我们发现招募到在HCV感染的脂滴可能代表亲或抗病毒宿主因子的蛋白质。我们的方法可以适用于各种不同的细胞和培养条件，如感染病原体，环境压力，或药物治疗。

脂滴是丙型肝炎病毒感染的病毒组装的推定位点是至关重要的，但形态和病毒颗粒的出口的分子机制知之甚少。为了鉴定参与该过程新颖的宿主依赖因素，我们进行了HCV感染的细胞21（ 图1A）的定量脂滴蛋白质组分析。我们建立了协议用于纯化脂滴，并定期检测的脂滴结合蛋白PLIN2 / ADRP和PLIN3 / TIP47和其他细胞区室的标记物，如β微管蛋白的对...

Watch this Scientific Journal Video about Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis at JoVE.com

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴是几种病原体，包括丙型肝炎病毒（HCV）复制重要的细胞器。我们描述分离为相关蛋白的定量质谱脂滴的方法;它可以在各种条件下，如病毒感染，环境压力，或药物治疗中使用。

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 ...

lipid-droplet-isolation-for-quantitative-mass-spectrometry-analysis

Research

JoVE Journal

Biochemistry

10.0K 次观看.  Leibniz Institute for Experimental Virology. 脂滴是几种病原体，包括丙型肝炎病毒（HCV）复制重要的细胞器。我们描述分离为相关蛋白的定量质谱脂滴的方法;它可以在各种条件下，如病毒感染，环境压力，或药物治疗中使用。 

视频: Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

科研

化学

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

生物工程

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

隔离细胞脂滴：两个纯化技术从酵母细胞与人类的胎盘开始

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

生物化学

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

生物学

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

啮齿动物组织的霰弹枪脂质组学

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

使用稳定同位素标记筛选脂质结构

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

作者聚焦：使用稳定同位素标记定量复杂脂质组学样品

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从 头 追踪脂质

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

脂滴隔离定量质谱分析

摘要

探索更多视频

此视频中的章节

隔离和脂质A的化学表征革兰氏阴性菌

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

隔离细胞脂滴：两个纯化技术从酵母细胞与人类的胎盘开始

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

一个定量评估的酵母使用电喷雾质谱法Lipidome

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

啮齿动物组织的霰弹枪脂质组学

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

脂滴隔离定量质谱分析

摘要

探索更多视频

此视频中的章节

隔离和脂质A的化学表征革兰氏阴性菌

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

隔离细胞脂滴：两个纯化技术从酵母细胞与人类的胎盘开始

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

一个定量评估的酵母使用电喷雾质谱法Lipidome

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

啮齿动物组织的霰弹枪脂质组学

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从 头 追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从 头 追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从 头 追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质

使用稳定同位素标记 LC-TIMS-TOF MS/MS 从头追踪脂质