Name: preparation of drosophila larval samples for gas chromatography-mass spectrometry (gc-ms)-based metabolomics
Uploaded: 2018-06-06T15:00:00
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...

<ol>
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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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:827px;" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Unsulfured blackstrap molasses</td>
			<td style="width:141px;">Good Food, INC</td>
			<td style="width:141px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td>Genesee Scientific</td>
			<td>66-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyridine</td>
			<td>EMD Millipore</td>
			<td>PX2012-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methoxyamine hydrocholoride (MOX)</td>
			<td>MP Biomedicals, LLC</td>
			<td>155405</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MSTFA with 1% trimethylchlorosilane</td>
			<td>Sigma</td>
			<td>69478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fleischmann&#8217;s Active dry yeast</td>
			<td>AB Mauri Food Inc</td>
			<td>2192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6oz Drosophila stock bottle</td>
			<td>Genesee Scientific</td>
			<td>32-130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soft tissue homogenizing mix (2 mL tubes)&#160;</td>
			<td>Omni International</td>
			<td>SKU:19-627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vial insert, 250 &#181;L deactivated glass with polymer feet</td>
			<td>Agilent</td>
			<td>5181-8872</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinic acid-2,2,3,3-d4</td>
			<td>Sigma</td>
			<td>293075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpeedVac</td>
			<td>Thermo&#160;</td>
			<td>SC210A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">o-Phosphoric acid</td>
			<td>Fisher Scientific</td>
			<td>A242-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propionic acid</td>
			<td>Sigma</td>
			<td>P5561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-Hydroxy benzoic acid methyl ester</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Ruptor</td>
			<td>Omni International</td>
			<td>SKU:19-040E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>5384000012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MultiTherm Shaker with a 24 X 12 mm block</td>
			<td>Benchmark Scientific</td>
			<td>H5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL centrifuge tube</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35 X 10 mm tissue culture dish</td>
			<td>Corning Incorporated</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

Research

JoVE Journal

Developmental Biology

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

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

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

Dissection of Larval Zebrafish Gonadal Tissue

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

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, ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Imaging Live Brain Explants from Drosophila melanogaster Third Instar Larvae

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants (Video) | JoVE

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging (Video) | JoVE 

解剖黑腹果蝇第三龄幼虫脑进行活细胞成像