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>

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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。我们强烈推荐这种方法进行定量脂滴蛋白质组分析。 由于富集或在MS分析中观察到脂滴相关蛋白的耗尽可能反映的感应或蛋白质表达的抑制，表达水平通过quantitativ分析ËRT-PCR和（优选地）western印迹建议。此外，两种方法可以被用来验证在脂滴的富集或特异性蛋白质的耗尽:脂滴分离后进行Western印迹，并用亲脂性染料，如BODIPY或LD540，即污点脂滴如所描述的21免疫荧光分析。 执行与交换标记条件的实验，以确保与"重"氨基酸标记不影响蛋白质表达或脂滴定位和从"轻"培养基和环境来源鉴定蛋白质污染物。蛋白质鉴定，搜索应与肽和蛋白水平二者0.01的假发现率（FDR）被执行。为确保高可靠的结果，我们建议进行至少3 - 4个独立的实验，从不同通道和不同的病毒股票的准备细胞。依赖于M变化的agnitude，可能需要更多的独立实验。 用于标准化定量MS数据以校正稍微不同的细胞数目或脂滴，通过所有鉴定的蛋白质的中值除以中心的"光"的检测率在"重"的肽，或反之亦然，在交换标记条件如所描述的25。如果脂滴的量的样品之间显著不同，标准化为脂滴的标记蛋白质，如PLIN2，可能是可取的。当我们归我们的MS数据PLIN2水平，我们发现了类似的结果，当我们向正常化的中位数（未显示分析）。值得注意的是，我们使用细胞培养条件下，我们并没有发现在HCV感染显著脂滴的积累。 脂滴在与其他细胞器，最显着的线粒体和ER亲密接触。因此，蛋白质˚FROM这些隔室可以与脂滴分离期间共分馏。如果这样的"污染物"蛋白丰不变响应感染或治疗，也不会影响比较脂滴蛋白质组分析。但是，必须指出的是，在某些情况下，脂滴及其他细胞器之间的接触可能会改变。例如，脂肪分解的条件下，线粒体蛋白质在从比在基础条件26 lipolytically刺激3T3-L1脂肪细胞中脂滴级分更高的频率检测到。刺激脂肪酸从头合成，另一方面，可能会导致与ER增强关联。这些变化则反映了各种刺激诱导的细胞器相互作用的变化和可能是有趣的，即使不反光纯脂滴定位。 我们利用这个协议大量的定量脂滴蛋白质组HCV感染的，相对于未感染的对照细胞电子分析揭示由HCV感染引起的扰动，并确定HCV复制21的调节剂。在肝癌细胞系的Huh7.5，我们经常鉴定到脂滴级分中的蛋白质2900，具有〜带在每个实验中的多个肽鉴定300克的蛋白质。继HCV感染的，我们观察到两个招聘和宿主蛋白的消耗。鉴定为在HCV感染的细胞的脂滴高度富集的几种蛋白质先前已公开为HCV宿主因素（ 例如，DEAD盒蛋白1和3（DDX1，DDX3）或胰岛素样生长因子-II mRNA的结合蛋白1（ IGF2BP1）），表明SILAC方法27，28，29的可靠性。此外，招募蛋白质通常为注释的RNA结合蛋白，突出病毒RNA replic的紧密结合通货膨胀与复合脂滴分数。相反，来自脂滴蛋白贫化主要注释脂质代谢过程，这表明HCV扰乱蛋白质组合物，以从它们的正常代谢调节和功能断开脂滴。 我们描述的协议是易于进行不同的细胞系和培养条件并能破译脂滴功能的依赖于脂滴复制不同病原体的生命周期有所帮助。

<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SILAC Protein Quantitation Kit - DMEM</td>
			<td>Thermo Fisher</td>
			<td>89983&#160;</td>
		</tr>
		<tr>
			<td>13C6 L-Arginine-HCl 50 mg</td>
			<td>Thermo Fisher</td>
			<td>88210&#160;</td>
		</tr>
		<tr>
			<td>Roti-Load 1</td>
			<td>Roth GmbH</td>
			<td>K929.1</td>
		</tr>
		<tr>
			<td>Roti-Blue 5x-Concentrate</td>
			<td>Roth GmbH</td>
			<td>A152.2&#160;</td>
		</tr>
		<tr>
			<td>10x SDS-Tris-Glycine - Buffer&#160;</td>
			<td>Geyer Th. GmbH &#38; Co.KG&#160;</td>
			<td>A1415,0250&#160;</td>
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		<tr>
			<td>GlutaMAX (100x)</td>
			<td>Life Technologies GmbH&#160;</td>
			<td>350500038&#160;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Solution for Cell Culture</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P4333-100ml&#160;</td>
		</tr>
		<tr>
			<td>PBS 1x Dulbeccos Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>D8537&#160;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>T3924-100ML&#160;</td>
		</tr>
		<tr>
			<td>Sodium Chloride BioChemica</td>
			<td>AppliChem GmbH</td>
			<td>A1149,1000</td>
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		<tr>
			<td>Tris Ultrapure&#160;&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A1086,5000A&#160;</td>
		</tr>
		<tr>
			<td>EDTA BioChemica</td>
			<td>AppliChem GmbH</td>
			<td>A1103,0250</td>
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		<tr>
			<td>Protease Inhibitor Cocktail 5ml</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P8340-5ML&#160;</td>
		</tr>
		<tr>
			<td>D(+)-Sucrose BioChemica</td>
			<td>AppliChem GmbH</td>
			<td>A3935,1000</td>
		</tr>
		<tr>
			<td>Hydrochloric acid 37% pure Ph. Eur., NF</td>
			<td>AppliChem GmbH</td>
			<td>A0625</td>
		</tr>
		<tr>
			<td>DC Protein Assay&#160;</td>
			<td>Bio-Rad Laboratoris GmbH&#160;</td>
			<td>500-0116&#160;</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem GmbH</td>
			<td>151339</td>
		</tr>
		<tr>
			<td>SDS Ultrapure</td>
			<td>AppliChem GmbH</td>
			<td>A1112</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>AppliChem GmbH</td>
			<td>A2331</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A4338</td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Invivogen</td>
			<td>ant-bl-1&#160;</td>
		</tr>
		<tr>
			<td>Potassium Chloride&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A1039</td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl Fluoride&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A0999</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>221309</td>
		</tr>
		<tr>
			<td>Dipotassium Hydrogenphosphate</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>P3786&#160;</td>
		</tr>
		<tr>
			<td>DTT&#160;</td>
			<td>AppliChem GmbH</td>
			<td>A2948</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>AppliChem</td>
			<td>A1694</td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>AppliChem</td>
			<td>A4974</td>
		</tr>
		<tr>
			<td>Nonfat dried milk powder</td>
			<td>AppliChem</td>
			<td>A0830</td>
		</tr>
		<tr>
			<td>Anti-ADFP/ ADRP&#160;</td>
			<td>abcam</td>
			<td>ab52355</td>
		</tr>
		<tr>
			<td>M6PRB1/TIP47 100&#181;g&#160;</td>
			<td>abcam</td>
			<td>ab47639</td>
		</tr>
		<tr>
			<td>Calreticulin, pAb 200 &#181;g</td>
			<td>Enzo Life Science GmbH&#160;</td>
			<td>ADI-SPA-600-F&#160;</td>
		</tr>
		<tr>
			<td>Anti-&#223;-Tubulin&#160;</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>T6074 200&#181;l &#160;</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Geyer Th. GmbH &#38; Co.KG&#160;</td>
			<td>A3678,0250 &#160;</td>
		</tr>
		<tr>
			<td>Anti-MnSOD</td>
			<td>Enzo Life Science GmbH&#160;</td>
			<td>ADI-SOD-110-F</td>
		</tr>
		<tr>
			<td>Anti-mouse HRP</td>
			<td>Thermo Fisher Pierce</td>
			<td>32430</td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP</td>
			<td>Thermo Fisher&#160; Pierce</td>
			<td>32460</td>
		</tr>
		<tr>
			<td>Amersham Hyperfilm ECL</td>
			<td>GE Healthcare</td>
			<td>28906836</td>
		</tr>
		<tr>
			<td>Lumi-Light Western Blotting Substrate</td>
			<td>Sigma-Aldrich Chemie GmbH&#160;</td>
			<td>12015196001</td>
		</tr>
		<tr>
			<td>96 Well Cell Culture Plate</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>655 180&#160;</td>
		</tr>
		<tr>
			<td>Terumo Syringe 1 ml</td>
			<td>Terumo</td>
			<td>SS-01T</td>
		</tr>
		<tr>
			<td>Filtropur BT 50, 500 ml, 0.45 &#181;m&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>83.1823.100&#160;</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN TGX Precast Gels, Any kD resolving gel</td>
			<td>Bio-Rad Laboratoris GmbH&#160;</td>
			<td>456-9034&#160;</td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>657160&#160;</td>
		</tr>
		<tr>
			<td>Dishes Nunclon 150/20&#160;</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>10098720 - 168381&#160;</td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>neoLab Migge GmbH&#160;</td>
			<td>C-8120&#160;</td>
		</tr>
		<tr>
			<td>Tube, 50 ml</td>
			<td>Greiner Bio-One GmbH&#160;</td>
			<td>227261&#160;</td>
		</tr>
		<tr>
			<td>SafeSeal Tube RNase-free&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>72.706.400&#160;</td>
		</tr>
		<tr>
			<td>Ultra Clear Centrifuge Tubes 11 x 60 mm</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>344062&#160;</td>
		</tr>
		<tr>
			<td>Suction Needles</td>
			<td>Transcodent</td>
			<td>6482</td>
		</tr>
		<tr>
			<td>Biosphere Fil. Tip 1000&#160;</td>
			<td>SARSTEDT&#160;</td>
			<td>70.762.211&#160;</td>
		</tr>
		<tr>
			<td>Biosphere Fil. Tip 200</td>
			<td>SARSTEDT&#160;</td>
			<td>70.760.211&#160;</td>
		</tr>
		<tr>
			<td>Biosphere Fil. Tip 10</td>
			<td>SARSTEDT&#160;</td>
			<td>70.1130.210&#160;</td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>11883722</td>
		</tr>
		<tr>
			<td>Pestles For Dounce All-Glass Tissue Grinders</td>
			<td>Fisher Scientific GmbH&#160;</td>
			<td>10389444</td>
		</tr>
		<tr>
			<td>Orbitrap Fusion</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Branson Sonifier 450</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer comfort, with Thermoblock 1.5 ml</td>
			<td>Eppendorf</td>
			<td>5355&#160;000.127</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module, and PowerPac Basic Power Supply,&#160;</td>
			<td>BioRad</td>
			<td>165-8033</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN 3 Multi-Casting Chamber</td>
			<td>BioRad</td>
			<td>165-4110</td>
		</tr>
		<tr>
			<td>PowerPac HC Power Supply</td>
			<td>Biorad</td>
			<td>164-5052</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424R</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>Optima L-90K</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>365670</td>
		</tr>
		<tr>
			<td>SW 60 Ti Rotor</td>
			<td>Beckman Coulter GmbH&#160;</td>
			<td>335649</td>
		</tr>
		<tr>
			<td>Infinite M1000 PRO</td>
			<td>Tecan</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微管蛋白的对微管的耗尽，MnSOD的线粒体的一个强有力的富集，或用于ER（ 图1B，C）钙网蛋白/钙联接蛋白。我们编译的可靠地与在Huh7.5细胞（ 图1D），脂滴蛋白cofractionate的列表，使用同位素标记，是专门招募鉴定的蛋白质到或从HCV感染的细胞（ 图1E）的脂滴位移。我们的研究结果表明，HCV从他们的常规代谢功能和/或法规断开脂滴及鉴定假定主机的依赖和制约因素。 <img alt="图1" src="/files/ftp_upload/55585/55585fig1.jpg" /> 图1: （A）实验方案。未经处理的Huh7.5细胞用"重"氨基酸或"光"的氨基酸。承载"光"氨基酸细胞感染的HCV病毒记者（JC1 NS5AB-mKO2-BSD）。 "轻"和"重"氨基酸标记的群体混合，并将脂滴由两个随后的超离心和三个洗涤步骤，通过SDS-PAGE分离分离，并且通过LC-ESI-MS / MS进行分析。注意ultracentrifugati后浮白脂滴分数（红色箭头）<html>上，并在微量离心管中洗涤。 （B）核后和脂滴分数的印迹分析显示脂滴标志蛋白的脂滴分数富集和其他细胞区室的标记的枯竭。后核子上清液（PNS），并通过SDS-PAGE分离的脂滴级分的考马斯蓝和银染色（C）。银染是提出了唯一的可视化。 （D）肽和Huh7.5细胞中的脂滴级分鉴定的蛋白质的蛋白质覆盖百分比的数量的热图（截止:≥5肽和≥20％覆盖率）。 （E）热图描绘富集或HCV感染后耗尽脂滴相关蛋白（归一化到平均，1.5倍截止，* P &lt;0.05，** P &lt;0.01，*** P &lt;0.001）。从21修改。等="_空白"&gt;点击此处查看该图的放大版本。

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

Nanyang Technological University

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

多肽基基质辅助激光解吸/电离质谱成像中福尔马林固定样品的优化制备工作流

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...