Name: preparation of drosophila larval samples for gas chromatography-mass spectrometry (gc-ms)-based metabolomics
Uploaded: 2018-06-06T15:00:00.000+00:00
Duration: 7 min 21 s
Description: Watch this Scientific Journal Video about Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics at JoVE.com

多亏了印第安纳大学的大众光谱学设施和犹他大学新陈代谢的核心设施来帮助优化这个协议。J.M.T. 由国立卫生研究院国立医学研究所 R35GM119557 奖编号支持。

1. 收集卵子
<ol><li>收集所需基因型的成年男性和处女女性。单独年龄这些动物在一个食物小瓶与标准布卢明顿媒介为3–5天。</li>
	<li>通过将50只处女母和25只雄性转移到一个新的食品瓶中, 建立适当的交配。 注: 每种基因型应至少设置六个独立交配。每种交配 (即从六个独立的交配收集的六个样本) 中只收集一个样本。</li>
	<li>
准备糖蜜下蛋帽。
	<ol>
<li>将115毫升糖蜜和29克琼脂与700毫升 H2O 在 2 L 瓶中混合。</li>
		<li>在热盘子上煮沸糖蜜琼脂混合物。</li>
		<li>冷却到70摄氏度。</li>
		<li>添加25毫升的酸混合 (20 毫升85% 磷酸和209毫升丙酸在 1 L 的 H2O;贮存在棕色瓶) 和10毫升10% 对羟基苯甲酸甲酯在95% 乙醇。</li>
		<li>将糖蜜琼脂倒入 35 x 10 毫米2组织培养皿的盖子和下半部分。 注: 在浇注糖蜜琼脂板之前, 确保 35 x 10 mm2板的盖子和/或底座适合 snuggly 到果蝇股票瓶 (步骤1.6 中描述) 的口中。根据使用的车牌品牌, 底座可能无法使用。</li>
	</ol>
</li>
	<li>将蒸馏水添加到活性干酵母中 (5 克 yeast/7 毫升水), 直到混合物变成糊状, 可以很容易地扩散到固体表面 (即, 花生黄油的一致性)。</li>
	<li>在糖蜜下蛋帽的表面上散布约1.5 克的酵母膏。</li>
	<li>用22克针戳四个空气孔到塑料6盎司的反面.果蝇股票瓶。</li>
	<li>将新交配的苍蝇从食物瓶转移到培养瓶中。 注: 苍蝇可以通过使用 CO2作为临时麻醉剂或通过保持瓶子的顶部, 并尖锐地敲击瓶子底部的台式。</li>
	<li>快速地放置一个糖蜜蛋盖与酵母糊在表面到一个果蝇瓶的嘴里。使用实验室胶带, 以确保产卵帽到位。 注: 如果苍蝇在转移前被麻醉, 应将瓶子水平放置在台式上, 直到所有苍蝇都恢复正常为止。一旦苍蝇是完全移动, 把瓶子放在一个孵化器与糖蜜产卵帽的文化底部。</li>
	<li>在头两天至少更换一次糖蜜蛋帽, 先反转股票瓶, 敲击瓶子底部的台式, 去掉旧的蛋帽, 然后立即将新的蛋帽放进瓶子的嘴里。扔掉旧的下蛋帽。 注意: 这一步确保雌性不持有卵子, 并允许收集同步种群。</li>
	<li>在3天后, 将一个新的下蛋帽放入库存瓶中, 在2小时后更换, 然后丢弃。在四小时后取出并更换第二个下蛋帽。用日期、时间和基因型标记这个下蛋帽的底部。 注: 在这4小时的窗口收集的鸡蛋将用于分析。此同步步骤对于分析特定的开发阶段至关重要。鸡蛋可以用这种方式收集好几天。</li>
	<li>将产卵帽插入 60 x 15 毫米2 Petri 板, 并将其置于理想的温度和湿度的孵化器中。</li>
	<li>每天检查产卵帽中的饥饿、人口密度或污染问题。 注意: 幼虫必须获得足够的食物, 以确保持续发展。如有必要, 新鲜的酵母膏可以在所有产卵帽上传播, 这将在实验中使用。此外, 如果幼虫种群密度过高, 幼虫就会开始从培养盘中游走。这些样本不应用于 metabolomic 分析, 因为高人口密度和中间爆发的饥饿经历, 而徘徊将导致数据不一致。建议每片80–100中二龄幼虫 (L2) 的密度为每片或40–50中第三龄幼虫 (L3)。应丢弃明显受细菌或真菌污染的样品。</li>
</ol>2. 幼虫标本采集
<ol><li>年龄幼虫直到理想的阶段。如果收集 L3 幼虫, 重新同步样品在 L2–L3 蜕皮。通过使用前面的气孔作为开发里程碑15的以前描述的方法, 通常实现重新同步。或者, mid-L3 幼虫可以使用sgs3报告基因16进行同步。 注: 在这方面, 我们发现 mid-L2 幼虫 (产卵后60小时) 最适合于大多数分析, 因为幼虫发育在这个阶段仍然比较同步, 仔细收集的样品有足够的食物发展到这个 timepoint, 和每样动物的数量是可管理的 (见下文)。另外一个同步步骤对于 L3 幼虫是必要的, 因为在 L3 开发过程中发生的大量昆虫蜕皮激素脉冲对新陈代谢产生了戏剧性的影响, 并且会在不同步的种群中生成工件。</li>
	<li>用解剖针轻轻地将酵母糊从琼脂上提起。把酵母放在一个新的糖蜜琼脂帽上。 注意: 大多数幼虫会在糖蜜琼脂和酵母膏之间的界面上进食。</li>
	<li>使用小画笔收集新暴露的糖蜜琼脂表面的幼虫。将幼虫放入冰上的1.5 毫升离心管中。 注: 每个发育阶段的适当样本大小如下:50 头龄幼虫 (L1);25 mid-L2 幼虫;20早期 L3 幼虫;15 mid-L3 或晚 L3 幼虫。</li>
	<li>在收集大样本集时, 在小批量 (四到六个样本) 中清洗和冻结样品 (步骤2.3 到 2.8)。样品必须在放置幼虫的20分钟内冷冻在试管中。 注: 我们建议使用离心1.5 毫升弹性管, 因为其他管可能会干扰在步骤3的样品转移。样品必须收集在离心管。不要收集步骤3.1 中描述的珠子管中的样品。陶瓷珠保留 NaCl 洗涤液, 导致质量测量不准确, 并将污染物引入样品中。</li>
	<li>将1毫升的冰冷0.9% 氯化钠加入管中, 关闭盖子, 垂直翻转管子彻底清洗幼虫。</li>
	<li>把管子放回冰上 ~ 三十年代。在这段时间里, 幼虫会沉到管子的底部, 但酵母会保持悬浮。一旦所有幼虫形成一个松散的颗粒, 去掉氯化钠溶液使用1毫升吸管。</li>
	<li>重复步骤2.4 和步骤2.5 两次, 或直到最终清洗解决方案是明确的。 注: 如果收集的样品含有过量的酵母, 可能需要额外的洗涤步骤。</li>
	<li>将样品在 2000 x g上离心1分钟, 摄氏4摄氏度。</li>
	<li>使用200µL 吸管去除所有残留溶液。</li>
	<li>立即将样品冷冻在液氮中。 注意: 在此步骤中可以暂停该协议。样品可储存3月至-80 摄氏度。</li>
</ol>3. 将样品转移到珠管上
<ol><li>每个幼虫样品必须从1.5 毫升离心管转移到2毫升 screwcap 管, 其中含有1.4 毫米陶瓷珠。为准备这一转移步骤, 使用一个无酒精标记标签的一侧2毫升 screwcap 珠管 (盖子上的标记将被销毁在步骤 4.4)。</li>
	<li>使用能够精确测量0.01 毫克的分析天平, 将标记的珠管的质量进行包装。</li>
	<li>如果幼虫样品储存在摄氏-80 摄氏度, 则在转移之前将样品管放在液氮中。</li>
	<li>使用长钳从液氮杜瓦中除去1.5 毫升的样品管。</li>
	<li>戴腈手套, 抓住冷冻管, 反转, 并大幅打击对台式的管盖, 以赶走冰冻颗粒。立即倒入一个预 tared 2 毫升 screwcap 珠管颗粒。如果球团无法取出, 则将管子回流至液氮并重复。 注意: 幼虫丸不会从所有离心管中去除。如果使用的不是推荐的管, 确定幼虫颗粒是否可以在收集样品之前脱落, 以进行分析。</li>
	<li>快速测定幼虫颗粒和珠管的组合质量。立即将样品管放入液氮中。幼虫颗粒质量将被用来规范化的 metabolomic 数据。 注意: 在此步骤中可以暂停该协议。样品可储存3月至-80 摄氏度。</li>
</ol>4. 样品提取
<ol><li>将样品管放置在-20 摄氏度台式冷却器中。</li>
	<li>添加0.8 毫升的 prechilled (-20 °c) 90% 甲醇含有2µg/毫升 succinic-d4 酸入每管。返回样品到-20 °c 台式冷却器。 注: succinic-d4 酸作为内部标准。仅使用 HPLC 级 H2O 和甲醇。由于甲醇和其他有机溶剂是挥发性的, 难以准确测量使用空气置换吸管, 我们建议使用正位移吸管的这一步骤和所有后续步骤。</li>
	<li>通过添加0.8 毫升的 prechilled (-20 °c) 90% 甲醇, 将含有2µg/毫升 succinic-d4 酸, 放入空珠管中, 建立负控制。</li>
	<li>融汇样品30秒在6.45 米/秒使用一个珠轧机均质机, 位于4°c 温度控制室。 注意: 未能迅速完全融汇幼虫颗粒是新陈代谢分析中常见的变异来源。只使用高压, 能够在三十年代内摧毁冰冻幼虫组织。</li>
	<li>将匀质样品管返回到-20 °c 台式冷却器, 并在-20 摄氏度冷藏库中孵化至少1小时。</li>
	<li>离心管在 2万 x g或最大速度为5分钟在4°c 去除结果沉淀。</li>
	<li>将上清的600µL 转化为新的1.5 毫升离心管。不要扰乱沉淀。如果沉淀颗粒在吹打上清时脱落, 返回所有上清到管并重复步骤4.6。</li>
	<li>将样品管放在真空离心机中。在密封离心机之前, 要确保所有管子都开着。在室温下干燥样品, 直到除去所有溶剂 (这个步骤通常需要16小时完成)。 注意: 如有必要, 可以在此步骤暂停协议。干燥样品可储存在-80 摄氏度。</li>
</ol>5. 化学衍生化
<ol><li>如果干燥样品储存在-80 摄氏度, 将未开封的样品管放在真空离心机中, 干燥30分钟。此步骤必须在打开取样管之前执行。 注意: 这一步去除从冷冻库中取出的离心管外的冷凝。协议的这一部分对 H2非常敏感, 必须采取额外的预防措施, 以使所有试剂尽可能干燥。</li>
	<li>
在无水吡啶中制备40毫克/毫升 methoxylamine 盐酸盐 (混合氧化物) 溶液。只使用无水吡啶。在干燥中储存混合氧化物和吡啶, 并每天准备混合氧化物溶液。 注意: 氧化物和吡啶是有毒的。在通风罩里准备这个溶液。<ol>
<li>用热枪干1毫升的玻璃注射器。</li>
		<li>将注射器针插入无水吡啶瓶中。除去1毫升吡啶, 加入含有40毫克混合氧化物的离心管。</li>
		<li>用氩气冲洗瓶无水吡啶。把瓶子密封起来, 回到干燥。</li>
		<li>在35摄氏度的热搅拌机中, 在600转每分钟10分钟内, 将混合氧化物溶解在吡啶中。</li>
	</ol>
</li>
	<li>在无水吡啶溶液中加入40µL 40 毫克/毫升混合氧化物的干燥样品。</li>
	<li>漩涡为十年代和简要离心机 (1万 x g二十年代)。</li>
	<li>在热混合器中以 600 rpm 在30摄氏度孵育1小时。</li>
	<li>离心机在 2万 x g或最大速度为5分钟去除微粒物质。</li>
	<li>将25µL 的清液转移到一个 autosampler 瓶中, 用250µL 停用的玻璃 microvolume 插入。</li>
	<li>添加40µL n-甲基-n-trimethylsilyltrifluoracetamide (MSTFA), 其中包含1% 差旅管理公司。 注意: MSTFA 是有毒的。在通风罩中执行此步骤。 注意: 如果可用, 此步骤可以由 Gerstel autosampler 完成。</li>
	<li>用卷发工具将瓶盖放在 autosampler 瓶上并密封。</li>
	<li>在37摄氏度孵育样品1小时, 晃动 (250 rpm)。</li>
	<li>准备一个脂肪酸甲酯标准 (FAMES) 解决方案, 以前 desceribed3。在注射前, 使用机器人 autosampler 在 autosampler 瓶中添加3µL FAMES。 注: FAMEs 用于校准固定时间移位并检查仪器性能。如果没有可用的机器人 autosampler, 则可以在步骤5.8 中添加 FAMEs。</li>
</ol>6. GC-MS 检测
注: 在大多数情况下, 用户将在一个质谱核心设施的协助下执行这一步骤。此协议设计用于与 30 m, GC 列与一个 5 m 卫兵专栏。
<ol><li>随机抽样顺序。</li>
	<li>
准备 GC-MS。
	<ol>
<li>将氦载流子气体流量设置为1毫升/分。</li>
		<li>将入口温度设置为250摄氏度。</li>
		<li>程序 GC 以执行以下温度渐变:<ol>
<li>初始温度到95°c 以保持1分钟。</li>
			<li>将温度提高到110摄氏度, 速度为40摄氏度/分钟, 保持2分钟。</li>
			<li>增加到摄氏250摄氏度/分钟坡道。</li>
			<li>增加到330°c 以25°c/分钟的速度, 最后举行4分钟。</li>
		</ol>
</li>
		<li>将溶剂延迟设置为3.5 分钟。 注: 根据 GC-MS 系统可以改变时间。这一步骤的目的是防止 MSTFA 和混合氧化物破坏探测器。</li>
	</ol>
</li>
	<li>将衍生物样品的1µL 注入 GC-MS (分离率为 10:1)。 注: 如果峰值强度过低, 则注入2µL。样品的注射顺序应该是随机的。</li>
	<li>在 50–500 m/z 的质量范围内, 在全扫描模式下操作质谱仪。</li>
</ol>7. 数据分析
<ol><li>使用目标或不相关方法分析 metabolomic 数据。目标分析的重点是测量一组定义的代谢物的丰度, 如下面我们描述的乳酸测量。相比之下, 不相关分析使用一种无偏见的方法来识别在两个样本集之间明显改变的任何代谢特征。 注意: 我们的实验室主要使用免费程序 MetAlign17和 MetaboAnalyst18,19进行数据分析。由于对质量控制、规范化和数据处理步骤的充分描述超出了本手稿的范围, 因此, 我们将用户推荐给用于数据处理20、21的更详细的协议, 22。此外, 还可以在8的其他地方找到描述此分析所需步骤的流程图。</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatography/mass+spectrometry+in+metabolic+profiling+of+biological+fluids.">Gas chromatography/mass spectrometry in metabolic profiling of biological fluids.</a> Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 871 (2), 202-211 (2008).</li><li>Want, E. J., Nordstrom, A., Morita, H., Siuzdak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+exogenous+to+endogenous:+The+inevitable+imprint+of+mass+spectrometry+in+metabolomics.">From exogenous to endogenous: The inevitable imprint of mass spectrometry in metabolomics.</a> Journal of Proteome Research. 6 (2), 459-468 (2007).</li><li>Garcia, A., Barbas, C., Metz, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metabolic Profiling: Methods and Protocols Vol. 708 Methods in Molecular Biology. , 191-204 (2011).</li><li>Chan, E. C. Y., Pasikanti, K. K., Nicholson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+urinary+metabolic+profiling+procedures+using+gas+chromatography-mass+spectrometry.">Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry.</a> Nature Protocols. 6 (10), 1483-1499 (2011).</li><li>Dunn, W. B., 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=Procedures+for+large-scale+metabolic+profiling+of+serum+and+plasma+using+gas+chromatography+and+liquid+chromatography+coupled+to+mass+spectrometry.">Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.</a> Nature Protocols. 6 (7), 1060-1083 (2011).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Laboratory Manual. , 171-178 (1989).</li><li>Biyasheva, A., Do, T. V., Lu, Y., Vaskova, M., Andres, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glue+secretion+in+the+Drosophila+salivary+gland:+a+model+for+steroid-regulated+exocytosis.">Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis.</a> Developmental Biology. 231 (1), 234-251 (2001).</li><li>Lommen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetAlign:+Interface-driven,+versatile+metabolomics+tool+for+hyphenated+full-scan+mass+spectrometry+data+preprocessing.">MetAlign: Interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing.</a> Analytical Chemistry. 81 (8), 3079-3086 (2009).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+MetaboAnalyst+3.0+for+comprehensive+metabolomics+data+analysis.">Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis.</a> Current Protocols in Bioinformatics. 55, (2016).</li><li>Xia, J., Sinelnikov, I. V., Han, B., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MetaboAnalyst+3.0-making+metabolomics+more+meaningful.">MetaboAnalyst 3.0-making metabolomics more meaningful.</a> Nucleic Acids Research. 43 (W1), W251-W257 (2015).</li><li>Lommen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+(pre-)processing+of+nominal+and+accurate+mass+LC-MS+or+GC-MS+data+using+MetAlign.">Data (pre-)processing of nominal and accurate mass LC-MS or GC-MS data using MetAlign.</a> Methods in Molecular Biology. 860, 229-253 (2012).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+MetaboAnalyst+3.0+for+comprehensive+metabolomics+data+analysis.">Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis.</a> Current Protocols in Bioinformatics. 55, (2016).</li><li>Xia, J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+inference+of+biological+patterns,+functions+and+pathways+from+metabolomic+data+using+MetaboAnalyst.">Web-based inference of biological patterns, functions and pathways from metabolomic data using MetaboAnalyst.</a> Nature Protocols. 6 (6), 743-760 (2011).</li><li>Li, H., Tennessen, 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=Methods+for+studying+the+metabolic+basis+of+Drosophila+development.">Methods for studying the metabolic basis of Drosophila development.</a> Wiley Interdisciplinary Reviews Developmental Biology. 6 (5), (2017).</li></ol>

作者没有什么可透露的。

新陈代谢提供了一个无与伦比的机会来调查代谢反应, 构成中间代谢。然而, 这种技术的敏感性使数据易受遗传背景、发育线索和各种环境应力的影响, 包括温度、湿度、人口密度和养分供应。因此, 高质量和可重复的新陈代谢分析要求在高度控制的条件下收集样品。虽然一些评论强调这一点3,8,23, 在这里我们提供了一个分步方?...

果蝇是一种研究调节中间代谢的分子机制的理想系统. 不仅是大多数代谢通路保存在果蝇和人类之间, 但关键的营养传感器和生长调节剂, 如胰岛素, Tor, 和 c-myc, 也活跃在飞1,2。因此,果蝇可以用来探索人类疾病的代谢基础, 从糖尿病和肥胖到变性和癌症。在这方面,果蝇幼虫的发育提供了一个理想的框架, 其中研究代谢计划称为有氧糖酵解, 或华宝效应。正如许多肿瘤使用有氧糖酵解产生碳水化合物的生物量, 所以做果蝇幼虫激活有氧糖酵解促进发展增长3,4,5。这些幼虫与肿瘤代谢的相似性建立了果蝇作为理解如何调节有氧糖酵解的关键模型体内。
尽管苍蝇已经成为研究新陈代谢的流行模型, 但大多数的果蝇研究依赖于用于测量单个代谢产物3(如海藻糖、甘油三酯或 ATP) 的方法。由于需要一个特定的协议来衡量每个代谢产物, 以化验为基础的研究是劳动密集型的, 昂贵的, 并偏向于那些可以用商业工具来衡量的化合物。这些限制的解决方案已经出现在新陈代谢领域, 它提供了研究果蝇新陈代谢的更有效和无偏见的方法。与基于检测的研究不同, 单 metabolomic 分析可以同时测量数以百计的小分子代谢物, 并全面了解生物体的新陈代谢状况6,7。这种技术大大扩大了果蝇代谢研究的范围, 并代表了这一新兴领域8的未来。
Metabolomic 研究主要采用三技术: (一) 核磁共振 (NMR), (ii.) 液相色谱-质谱 (LC), (iii.) 气相色谱-质谱 (gc-ms)9。每种方法都具有明显的优缺点, 所有这些技术都已用于成功研究果蝇代谢。由于我们实验室进行的研究集中在小的极性代谢物上, 我们主要采用了基于 GC-MS 的方法。GC-MS 为用户提供了许多优点, 包括高重现性, 峰值分辨率, 灵敏度, 以及标准电子撞击 (EI) 光谱库的有效性, 它允许快速识别发现的代谢功能10,11。但是, GC-MS 的样品的制备有点复杂, 需要仔细注意细节。样品必须收集, 洗涤, 称量和冷冻的方式, 迅速止渴代谢反应。此外, 苍蝇胴体对标准均匀化协议有抗药性, 需要一个珠磨来确保最佳的代谢产物萃取。最后, 在检测12之前, 用 GC-MS 分析的样品必须经过化学衍生。虽然以前发布的方法描述了所有这些步骤3、13、14, 但仍然需要一个允许新手用户重现性生成高质量数据的可视化协议。在这里, 我们演示如何准备果蝇幼虫样品的 GC-MS 新陈代谢分析。该协议允许用户重现性测量许多组成中央碳代谢的小极性代谢物。

<table><tbody><tr><td>Unsulfured blackstrap molasses</td><td>Good Food, INC</td><td></td><td></td></tr><tr><td>Drosophila Agar Type II</td><td>Genesee Scientific</td><td>66-103</td><td></td></tr><tr><td>Pyridine</td><td>EMD Millipore</td><td>PX2012-7</td><td></td></tr><tr><td>Methoxyamine hydrocholoride (MOX)</td><td>MP Biomedicals, LLC</td><td>155405</td><td></td></tr><tr><td>MSTFA with 1% trimethylchlorosilane</td><td>Sigma</td><td>69478</td><td></td></tr><tr><td>Fleischmann&amp;rsquo;s Active dry yeast</td><td>AB Mauri Food Inc</td><td>2192</td><td></td></tr><tr><td>6oz Drosophila stock bottle</td><td>Genesee Scientific</td><td>32-130</td><td></td></tr><tr><td>Soft tissue homogenizing mix (2 mL tubes)&amp;nbsp;</td><td>Omni International</td><td>SKU:19-627</td><td></td></tr><tr><td>Vial insert, 250 &amp;micro;L deactivated glass with polymer feet</td><td>Agilent</td><td>5181-8872</td><td></td></tr><tr><td>Succinic acid-2,2,3,3-d&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>293075</td><td></td></tr><tr><td>SpeedVac</td><td>Thermo&amp;nbsp;</td><td>SC210A</td><td></td></tr><tr><td>o-Phosphoric acid</td><td>Fisher Scientific</td><td>A242-1</td><td></td></tr><tr><td>Propionic acid</td><td>Sigma</td><td>P5561</td><td></td></tr><tr><td>p-Hydroxy benzoic acid methyl ester</td><td>Genesee Scientific</td><td>20-258</td><td></td></tr><tr><td>Bead Ruptor</td><td>Omni International</td><td>SKU:19-040E</td><td></td></tr><tr><td>ThermoMixer F1.5</td><td>Eppendorf</td><td>5384000012</td><td></td></tr><tr><td>MultiTherm Shaker with a 24 X 12 mm block</td><td>Benchmark Scientific</td><td>H5000</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td></td></tr><tr><td>1.5 mL centrifuge tube</td><td>Eppendorf</td><td>22364111</td><td></td></tr><tr><td>Falcon 35 X 10 mm tissue culture dish</td><td>Corning Incorporated</td><td>353001</td><td></td></tr><tr><td>GC column</td><td>Phenomex</td><td>ZB-5MSi</td><td></td></tr></tbody></table>

preparation of drosophila larval samples for gas chromatography-mass spectrometry (gc-ms)-based metabolomics

最近在新陈代谢领域的进展已建立了果蝇的果蝇, 作为研究动物代谢的强大遗传模型. 通过将大量的果蝇基因工具与调查大量中间代谢的能力结合起来, 新陈代谢方法可以揭示饮食、基因型、生命史事件和环境线索之间的复杂相互作用。此外, 新陈代谢研究可以发现新的酶机制, 并揭示以前未知的联系之间似乎完全不同的代谢途径。为了便于在果蝇社区中更广泛地使用此技术, 我们在这里提供了一个详细的协议, 描述如何准备用于气相色谱-质谱 (GC-MS) 的果蝇幼虫样品-基于 metabolomic 分析。我们的协议包括幼虫样本采集、代谢物提取、化学衍生和 GC-MS 分析的描述。该协议的成功完成将使用户能够测量小极性代谢物的相对丰度, 包括氨基酸、糖和糖酵解和 TCA 循环所涉及的有机酸。

乳酸脱氢酶 (dLDH) 突变体缺乏 dLDH 活性 4, 基因匹配控制被收集为 mid-L2 幼虫, 并按照上文所述的协议进行处理。与对照组相比, 突变幼虫在乳酸、丙酮酸和 L-2-hydroxyglutarate4中表现出显著变化。光谱是通过安捷伦 GC6890-5973i MS 系统获得的。使用我们的协议生成的 GC-MS 频谱示例显示在图 1中。海藻糖有许多可见的?...

Watch this Scientific Journal Video about Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics at JoVE.com

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

此协议描述如何准备果蝇幼虫进行基于 GC-MS 的 metabolomic 分析。

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

preparation-drosophila-larval-samples-for-gas-chromatography-mass

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

11.1K 次观看.  Indiana University. 此协议描述如何准备果蝇幼虫进行基于 GC-MS 的 metabolomic 分析。

视频: Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

在代谢分析准备线粒体富集级的<em&gt;果蝇</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

科研

发育生物学

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

遗传学

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

中等规模的制备<em&gt;果蝇</em&gt;蛋白质组学实验胚胎提取物

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

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 ...

生物化学

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

神经科学

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

生物学

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

使用 FRET 传感器监测 果蝇 中的脑代谢物运输

Visualizing Monocarboxylates and Other Relevant Metabolites in the Ex Vivo Drosophila Larval Brain Using Genetically Encoded Sensors

The high energy requirements of brains due to electrical activity are one of their most distinguishing features. These requirements are met by the production of ATP from glucose and its metabolites, such as the monocarboxylates lactate and pyruvate. It is still unclear how this process is regulated or who the key players are, particularly in Drosophila.
Using genetically encoded F&#246;rster resonance energy transfer-based sensors, we present a simple method for measuring the transport of monocarboxylates and glucose in glial cells and neurons in an ex-vivo Drosophila larval brain preparation. The protocol describes how to dissect and adhere a larval brain expressing one of the sensors to a glass coverslip. 
We present the results of an entire experiment in which lactate transport was measured in larval brains by knocking down previously identified monocarboxylate transporters in glial cells. Furthermore, we demonstrate how to rapidly increase neuronal activity and track metabolite changes in the active brain. The described method, which provides all necessary information, can be used to analyze other Drosophila living tissues.

作者聚焦:使用 果蝇 模型的脑能量代谢研究进展

Here we present a protocol to visualize the transport of monocarboxylates, glucose, and ATP in glial cells and neurons using genetically encoded F&#246;rster resonance energy transfer-based sensors in an ex-vivo Drosophila larval brain preparation.

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

摘要

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此视频中的章节

在代谢分析准备线粒体富集级的

全基因组对真核生物转录误差的监测

中等规模的制备

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

果蝇幼虫和成年脑的代谢分析

成绩单和代谢产物的分离纯化

优化果蝇幼虫神经肌肉接头透射电子显微镜样品制备工艺

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

摘要

探索更多视频

此视频中的章节

在代谢分析准备线粒体富集级的

全基因组对真核生物转录误差的监测

中等规模的制备

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

果蝇幼虫和成年脑的代谢分析

成绩单和代谢产物的分离纯化

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在 离体果蝇 幼虫大脑中可视化单羧酸盐和其他相关代谢物

优化果蝇幼虫神经肌肉接头透射电子显微镜样品制备工艺

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物

使用基因编码传感器在离体果蝇幼虫大脑中可视化单羧酸盐和其他相关代谢物