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

Tak til medlemmerne af Indiana University masse spektroskopi facilitet og University of Utah Metabolomics Core facilitet for hjælp til optimering af denne protokol. J.M.T. er støttet af National Institute of General Medical Sciences af National Institutes of Health under Award nummer R35GM119557.

1. ægudtagning
<ol><li>Indsamle voksne hanner og jomfruelige hunner af de ønskede genotyper. Individuelt alder disse dyr i en mad hætteglas med standard Bloomington medier for 3-5 dage.</li>
	<li>Oprettet den passende parringer ved at overføre 50 jomfruelige hunner og 25 mænd til en ny mad hætteglas. Bemærk: Et minimum af seks uafhængige parringer bør sættes op for hver genotype. Kun én prøve vil blive indsamlet fra hver parring (dvs. seks prøver indsamlet fra seks uafhængige parringer).</li>
	<li>
<st...

<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=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68 (1), 105-115 (2014).</li><li>Li, H., 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=Drosophila+larvae+synthesize+the+putative+oncometabolite+L-2-hydroxyglutarate+during+normal+developmental+growth.">Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (6), 1353-1358 (2017).</li><li>Tennessen, J. M., Baker, K. D., Lam, G., Evans, J., Thummel, C. 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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. 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Metabolomics giver en enestående mulighed for at undersøge den metaboliske reaktioner, at komponerer formidlende stofskifte. Følsomheden af denne teknologi, men gør data modtagelige for genetiske baggrund, udviklingsmæssige stikord og en række forskellige miljøbelastninger, herunder temperatur, luftfugtighed, befolkningstæthed og næringsstoftilgængelighed. Derfor, en høj kvalitet og reproducerbare metabolomics analyse kræver, at prøver indsamles under stærkt kontrollerede forhold. Mens flere anmeldelser fre...

Bananfluen Drosophila melanogaster fremstod som et ideelt system til at studere den molekylære mekanisme, der regulerer formidlende stofskifte. Ikke kun bevares de fleste stofskifteveje mellem Drosophila og mennesker, men nøglen næringsstof sensorer og vækstregulerende som insulin, Tor og myc, er også aktiv i at flyve1,2. Som et resultat, kan Drosophila bruges til at udforske det metaboliske grundlaget for menneskelige sygdomme lige fra diabetes og fedme til neurodegeneration og kræft. I denne henseende giver Drosophila larve udvikling den ideelle ramme at studere en metabolisk program kendt som aerob glykolyse eller Warburg-effekten. Ligesom mange tumorer bruge aerob glykolyse til at generere biomasse fra kulhydrater, så aktivere for at gøre Drosophila larver aerob glykolyse for at fremme udviklingsmæssige vækst3,4,5. Disse ligheder mellem larve og tumor stofskifte etablere Drosophila som en vigtig model for forståelse hvordan aerob glykolyse er reguleret i vivo.
Trods det faktum, at fluen er opstået som en populær model til at studere metabolisme, stole de fleste Drosophila undersøgelser på metoder, der er designet til at måle individuelle metabolitter3, såsom trehalose, triglycerider, eller ATP. Da en bestemt protokol er forpligtet til at måle hver metabolit, er assay-baserede undersøgelser arbejdskrævende, dyrt og forudindtaget over for disse forbindelser, der kan måles ved hjælp af kommercielle kits. En løsning på disse begrænsninger er opstået fra feltet af metabolomics, som giver en mere effektiv og fordomsfri middel til at studere Drosophila stofskifte. I modsætning til en assay-baseret undersøgelse, kan en enkelt metabolomic analyse samtidig måle hundredvis af små molekyle metabolitter og giver en omfattende forståelse af en organisme metaboliske status6,7. Denne teknik er betydeligt udvidet anvendelsesområdet for Drosophila metaboliske undersøgelser og repræsenterer fremtiden for dette nye felt8.
Metabolomic undersøgelser er primært gennemført ved hjælp af tre teknologier: a Kernemagnetisk resonans (NMR), (ii) liquid chromatography-massespektrometri (LC-MS) og (iii) gaskromatografi-massespektrometri (GC-MS)9. Hver metode giver forskellige fordele og ulemper, og alle disse teknologier har været vant til med held studere Drosophila stofskifte. Da den forskning, som udføres i vores lab er fokuseret på små, polar metabolitter, beskæftiger vi primært en GC-MS-baseret metode. GC-MS giver brugeren en række fordele, herunder høj reproducerbarhed, peak opløsning, følsomhed, og tilgængeligheden af en standard elektron indvirkning (EI) spektrale bibliotek, som giver mulighed for hurtig identifikation af opdagede metaboliske funktioner10,11. Forberedelse af prøver for GC-MS, men er noget kompliceret og kræver en omhyggelig opmærksomhed for detaljer. Prøverne skal indsamles, vasket, vejes, og frosset i en måde, der hurtigt slukker metaboliske reaktioner. Desuden flyve slagtekroppen er resistente over for standard homogenisering protokoller og kræver en perle mølle til at sikre optimal metabolit udvinding. Endelig skal prøver analyseres ved GC-MS gennemgå kemiske forædling inden registrering af12. Mens tidligere udgivne metoder beskrive alle disse trin3,13,14, er en visuel protokol, der ville tillade begynderbrugerens reproducerbar generere data af høj kvalitet stadig nødvendig. Her viser vi hvordan du forbereder Drosophila larve prøver for GC-MS-baseret metabolomics analyse. Denne protokol tillader brugeren at reproducerbar måling af mange af de små polar metabolitter, at komponerer central carbon stofskifte.

<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

De seneste fremskridt inden for metabolomics har etableret bananfluen Drosophila melanogaster som en kraftfuld genetiske model til at studere dyrenes stofskifte. Ved at kombinere det enorme opbud af Drosophila genetiske værktøjer med evnen til at undersøge store skår af formidlende stofskifte, kan en metabolomics tilgang afsløre komplekse interaktioner mellem kost, genotype, livshistorie begivenheder og miljømæssige stikord. Derudover kan metabolomics undersøgelser opdage roman enzymatisk mekanismer og afdække hidtil ukendte forbindelser mellem tilsyneladende forskellige stofskifteveje. For at lette mere udbredt brug af denne teknologi blandt Fællesskabets Drosophila , her vi leverer en detaljeret protokol, der beskriver, hvordan du forbereder Drosophila larve prøver til gaskromatografi-massespektrometri (GC-MS)- baseret metabolomic analyse. Vores protokol indeholder beskrivelser af larve prøvetagning, metabolit udvinding, kemiske forædling og GC-MS analyse. Vellykket gennemførelse af denne protokol vil tillade brugere at måle den relative forekomst af små polar metabolitter, herunder aminosyrer, sukker og organiske syrer involveret i glycolysen og TCA cyklus.

Laktat dehydrogenase (dLDH) mutanter, som mangler dLDH aktivitet4og genetisk-matchede kontrol blev indsamlet som midten af L2 larver og forarbejdet i henhold til protokollen beskrevet ovenfor. Sammenlignet med kontrol, udviser mutant larver betydelige ændringer i laktat, pyruvat, og L-2-hydroxyglutarate4. Spectra er anskaffet med en Agilent GC6890-5973i MS system. Et eksempel på GC-MS-spektre generere med vores protokol er vist i ...

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

Denne protokol beskriver hvordan man forbereder Drosophila larver for GC-MS-baseret metabolomic analyse.

Forberedelse af Drosophila larve prøver til gaskromatografi-massespektrometri (GC-MS)-baseret 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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Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

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Drosophila Embryo Forberedelse og Microinjection for Levende Cell Mikroskopi Udført ved hjælp af en automatiseret højt indhold 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 (Video) | JoVE

Live-celle billeddannelse af Drosophila melanogaster tredje stjerne larvehjerner

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 

Dissektion af Drosophila melanogaster tredje stjerne larvehjerne til levende cellebilleddannelse