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

Thanks to members of the Indiana University Mass Spectroscopy Facility and the University of Utah Metabolomics Core Facility for assistance in optimizing this protocol. J.M.T. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM119557.

1. Egg Collection
<ol>
	<li>Collect adult males and virgin females of the desired genotypes. Individually age these animals in a food vial with standard Bloomington media for 3&#8211;5 days.</li>
	<li>Set up the appropriate matings by transferring 50 virgin females and 25 males to a new food vial. 
	NOTE: A minimum of six independent matings should be set up for each genotype. Only one sample will be collected from each mating (i.e., six samples collected from six independent matings).</li>
	<li>Prepare mo...

<ol>
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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. 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Metabolomics provides an unparalleled opportunity to survey the metabolic reactions that compose intermediary metabolism. The sensitivity of this technology, however, renders data susceptible to genetic background, developmental cues, and a variety of environmental stresses, including temperature, humidity, population density, and nutrient availability. Therefore, a high quality and reproducible metabolomics analysis requires that samples be collected under highly controlled conditions. While several reviews emphasize th...

The fruit fly Drosophila melanogaster has emerged as an ideal system for studying the molecular mechanism that regulate intermediary metabolism. Not only are most metabolic pathways conserved between Drosophila and humans, but key nutrient sensors and growth regulators, such as insulin, Tor, and myc, are also active in the fly1,2. As a result, Drosophila can be used to explore the metabolic basis of human diseases ranging from diabetes and obesity to neurodegeneration and cancer. In this regard, Drosophila larval development provides the ideal framework in which to study a metabolic program known as aerobic glycolysis, or the Warburg effect. Just as many tumors use aerobic glycolysis to generate biomass from carbohydrates, so to do Drosophila larvae activate aerobic glycolysis to promote developmental growth3,4,5. These similarities between larval and tumor metabolism establish Drosophila as a key model for understanding how aerobic glycolysis is regulated in vivo.
Despite the fact that the fly has emerged as a popular model for studying metabolism, most Drosophila studies rely on methods that are designed to measure individual metabolites3, such as trehalose, triglycerides, or ATP. Since a specific protocol is required to measure each metabolite, assay-based studies are labor-intensive, expensive, and biased towards those compounds that can be measured using commercial kits. A solution to these limitations has emerged from the field of metabolomics, which provides a more efficient and unbiased means of studying Drosophila metabolism. Unlike an assay-based study, a single metabolomic analysis can simultaneously measure hundreds of small molecule metabolites and provide a comprehensive understanding of an organism&#39;s metabolic status6,7. This technique has significantly expanded the scope of Drosophila metabolic studies and represents the future of this emerging field8.
Metabolomic studies are primarily conducted using three technologies: (i) nuclear magnetic resonance (NMR), (ii) liquid chromatography-mass spectrometry (LC-MS), and (iii) gas chromatography-mass spectrometry (GC-MS)9. Each approach offers distinct advantages and disadvantages, and all of these technologies have been used to successfully study Drosophila metabolism. Since the research conducted in our lab is focused on small, polar metabolites, we primarily employ a GC-MS-based method. GC-MS provides the user with a number of advantages, including high reproducibility, peak resolution, sensitivity, and the availability of a standard electron impact (EI) spectral library, which allows for the rapid identification of discovered metabolic features10,11. The preparation of samples for GC-MS, however, is somewhat complex and requires a careful attention to detail. Samples must be collected, washed, weighed, and frozen in a manner that quickly quenches metabolic reactions. Furthermore, the fly carcass is resistant to standard homogenization protocols and requires a bead mill to ensure optimal metabolite extraction. Finally, samples analyzed by GC-MS must undergo chemical derivatization prior to detection12. While previously published methods describe all of these steps3,13,14, a visual protocol that would allow the novice user to reproducibly generate high quality data is still needed. Here we demonstrate how to prepare Drosophila larval samples for GC-MS-based metabolomics analysis. This protocol allows the user to reproducibly measure many of the small polar metabolites that compose central carbon metabolism.

<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

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Lactate dehydrogenase (dLDH) mutants, which lack dLDH activity4, and genetically-matched controls were collected as mid-L2 larvae and processed according to protocol described above. When compared with controls, mutant larvae exhibit significant changes in lactate, pyruvate, and L-2-hydroxyglutarate4. Spectra were acquired with an Agilent GC6890-5973i MS system. An example of the GC-MS spectra generate with our protocol is shown in ...

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

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

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

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

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