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 ...

This method can answer key questions in the metabolism field, such as how the oncometabolite L2-hydroxyglutarate is synthesized in healthy cells. The main advantage of this technique is that it allows the user to directly measure over 100 metabolites in a sensitive and reproducible manner. Generally, individuals new to this method will struggle because the protocol requires efficient sample processing and is extremely sensitive to water vapor.To begin the protocol, add one milliliter of ice-cold 0.9%sodium chloride into a 1.5-milliliter tube containing previously prepared larvae. Close the lid, vertically flip the tube to thoroughly wash the larvae, and place the tube on ice for 30 seconds. The larvae will sink to the bottom of the tube, but the yeast will remain in suspension.Once all larvae have formed a loose pellet, remove the sodium chloride solution using a one-milliliter pipette. Centrifuge the samples at 2, 000 g for one minute at four degrees Celsius. Remove all residual solution using a 200-microliter pipette, and immediately freeze the sample in liquid nitrogen.Use an ethanol-proof marker to label the side of a two-milliliter screw-cap bead tube. Next, tare the mass of the labeled bead tube using an analytical balance capable of accurately measuring 0.01 milligrams. Use long forceps to remove the 1.5-milliliter sample tube from the liquid nitrogen dewar.Wearing nitrile gloves, grab the frozen tube, invert it, and sharply pound the tube lid against the benchtop to dislodge the frozen pellet. Immediately pour the pellet into a pre-tared two-milliliter screw-cap bead tube. The sample must be quickly processed to ensure that endogenous metabolic pathways are not reactivated.Quickly measure the combined mass of the larval pellet and bead tube. Immediately place the sample tube into liquid nitrogen. Place the sample tubes in a negative 20 degree Celsius benchtop cooler.Add 0.8 milliliters of pre-chilled 90%methanol containing two micrograms per milliliter of succinic-D4 acid into each tube. Return the samples to the negative 20 degree Celsius benchtop cooler. Set up a negative control by adding 0.8 milliliters of pre-chilled 90%methanol containing two micrograms per milliliter of succinic-D4 acid into an empty bead tube.Then, homogenize the samples for 30 seconds at 6.45 meters per second using a bead mill homogenizer located in a four degree Celsius temperature control room. Return the homogenized samples to the negative 20 degree Celsius benchtop cooler, and transfer the cooler into a negative 20 degree Celsius freezer for at least one hour. After incubating, centrifuge the tubes at 20, 000 g for five minutes at four degrees Celsius to remove the resulting precipitate.Transfer 600 microliters of the supernatant into a new 1.5-milliliter microcentrifuge tube, and do not disturb the precipitate. Open all the sample tubes, and place them in a vacuum centrifuge. Dry the samples at room temperature until all solvent is removed.If the dried samples were stored at negative 80 degrees Celsius, place unopened sample tubes in a vacuum centrifuge and dry them for 30 minutes. Prepare a solution of 40 milligrams per milliliter methoxylamine hydrochloride, or MOX, in anhydrous pyridine. Store the MOX and pyridine in a desiccator.Next, use a heat gun to dry a one-milliliter glass syringe for about five seconds. Insert the needle of the syringe into the bottle of anhydrous pyridine. Remove one milliliter of pyridine, and add it to a microfuge tube containing 40 milligrams of MOX.This portion of the protocol is extremely sensitive to water. All reagents must be stored in a desiccator prior to use. In addition, the user must ensure that the sample has minimal exposure to atmospheric water vapor.Then, flush the bottle of anhydrous pyridine with argon. Seal the bottle, and return it to the desiccator. Dissolve the MOX in pyridine by incubating the tube in a thermal mixer at 35 degrees Celsius for 10 minutes at 600 rpm.Next, add 40 microliters of 40 milligrams per milliliter MOX in anhydrous pyridine solution to the dried sample. Vortex the sample for 10 seconds, and briefly centrifuge it. Then, incubate the sample in a thermal mixer.Centrifuge the sample at 20, 000 g or maximum speed for five minutes to remove the particle matter. Transfer 25 microliters of the supernatant into an autosampler vial with a 250-microliter deactivated glass microvolume insert. Add 40 microliters of MSTFA containing 1%TMCS.Place a cap on the autosampler vial, and seal the vial using a crimper tool. Incubate the sample at 37 degrees Celsius for one hour with shaking at 250 rpm. Finally, add three microliters of previously prepared fatty acid methyl ester standard, or FAMES, solution, to the autosampler vial using a robotic autosampler immediately before injection.Gas chromatography-mass spectrometry of lactate dehydrogenase mutant larvae exhibit significant changes in lactate, pyruvate, and L2-hydroxyglutarate compared with control larvae. The GC-MS spectra generated with the protocol show many visible features and a notable peak for trehalose, which normally represents the largest peak in a larval sample. Principle component analysis, or PCA, clearly shows that the knockout and wild-type groups separate from each other and that there is no outlier in either group.While attempting this procedure, it's important to remember to keep the samples frozen prior to homogenization and to keep all reagents dry throughout the procedure.

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The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

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

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 

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