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

Takk til medlemmer av Indiana University masse Spectroscopy anlegget og University of Utah Metabolomics Core anlegg for hjelp til å optimalisere denne protokollen. J.M.T. støttes av National Institute of General Medical Sciences av National Institutes of Health under prisen nummer R35GM119557.

1. høsting
<ol><li>Samle voksne menn og virgin kvinner av de ønskede genotyper. Individuelt alder disse dyrene i mat ampuller med standard Bloomington medier for 3-5 dager.</li>
	<li>Angi den aktuelle parring ved å overføre 50 jomfru kvinner og 25 menn til en ny mat medisinglass. Merk: Minimum seks uavhengige parring bør settes opp for hver genotype. Bare ett eksempel hentes fra hver mating (dvs. seks prøver innhentet fra seks uavhengige parring).</li>
	<li>
Forberede melasse egg-legging caps....

<ol>
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C., Holmes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabonomics':+understanding+the+metabolic+responses+of+living+systems+to+pathophysiological+stimuli+via+multivariate+statistical+analysis+of+biological+NMR+spectroscopic+data.">Metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data.</a> Xenobiotica. 29 (11), 1181-1189 (1999).</li><li>Fiehn, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+-+the+link+between+genotypes+and+phenotypes.">Metabolomics - the link between genotypes and phenotypes.</a> Plant Molecular Biology. 48 (1-2), 155-171 (2002).</li><li>Cox, J. E., Thummel, C. S., 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=Metabolomic+Studies+in+Drosophila.">Metabolomic Studies in Drosophila.</a> Genetics. 206 (3), 1169-1185 (2017).</li><li>Lenz, E. M., Wilson, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+strategies+in+metabonomics.">Analytical strategies in metabonomics.</a> Journal of Proteome Research. 6 (2), 443-458 (2007).</li><li>Pasikanti, K. K., Ho, P. C., Chan, E. C. 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 gir en enestående mulighet å kartlegge metabolske reaksjoner som utgjør mellomledd metabolisme. Følsomheten av denne teknologien, men gjengir data utsatt for genetisk bakgrunn, utviklingsmessige signaler og en rekke miljømessige påkjenninger, inkludert temperatur, fuktighet, befolkningstetthet og næringsstoffer tilgjengelighet. Derfor en høy kvalitet og reproduserbar metabolomics analyse krever at prøver samles under kontrollerte forhold. Mens flere anmeldelser understreke dette poenget<sup class="x...

Frukt fly Drosophila melanogaster har dukket opp som et ideelt system for å studere molekylære mekanismen som regulerer mellomledd metabolisme. Ikke bare er de fleste metabolske veier konservert mellom Drosophila og mennesker, men viktig næringsstoff sensorer og vekst regulatorer, som insulin, Tor og myc, er også aktive i fly1,2. Resultatet kan Drosophila brukes å utforske metabolske grunnlaget for menneskelige sykdommer spenner fra diabetes og fedme neurodegeneration og kreft. I denne forbindelse gir Drosophila larver utvikling den ideelle rammen i å studere et metabolske program kalt aerobe Glykolysen eller Warburg effekten. Like mange svulster bruker aerobic Glykolysen for å generere biomasse fra karbohydrater, så aktivere gjør Drosophila Larvene aerobic Glykolysen å fremme utviklingsmessige vekst3,4,5. Disse likhetene mellom larver og svulst metabolisme etablere Drosophila som en viktig modell for å forstå hvordan aerobic Glykolysen er regulert i vivo.
Til tross for det faktum at fly har dukket opp som en populær modell for å studere metabolisme, avhengige de fleste Drosophila studier av metodene som er utviklet for å måle personlige metabolitter3, som trehalose, triglyserider eller ATP. Siden en bestemt protokoll er nødvendig å måle hver metabolitten, er analysen-baserte studier arbeidskrevende, dyrt og partisk mot de forbindelsene som kan måles med kommersielle kits. En løsning på disse begrensningene har dukket opp fra feltet i metabolomics, som gir en mer effektiv og saklig måte å studere Drosophila metabolisme. I motsetning til en analysen-basert studie, kan en enkelt metabolomic analyse samtidig måle hundrevis av små molekyl metabolitter og gir en omfattende forståelse av en organismes metabolske status6,7. Denne teknikken har betydelig utvidet omfanget av Drosophila metabolske studier og representerer fremtiden for denne nye feltet8.
Metabolomic studier er hovedsakelig utført med tre teknologier: (i) kjernefysiske magnetisk resonans (NRM), (ii) flytende kromatografi-massespektrometri (LC-MS) og (iii) gass kromatografi-massespektrometri (GC-MS)9. Hver tilnærming tilbyr forskjellige fordeler og ulemper, og alle disse teknologiene har blitt brukt til å kunne studere Drosophila metabolisme. Siden forskning utført i vår lab er fokusert på små, polar metabolitter, bruker vi primært en GC-MS-basert metode. GC-MS gir brukeren en rekke fordeler, inkludert høy reproduserbarhet, høyeste oppløsning, følsomhet og tilgjengeligheten av et standard elektron innvirkning (EI) spectral bibliotek, som gir mulighet for rask identifisering av oppdaget metabolske funksjoner10,11. Utarbeidelse av prøver for GC-MS, men er noe komplisert og krever en spesiell oppmerksomhet for detaljer. Prøver må være samlet, vasket, veide og frosset på en måte som raskt slukker metabolske reaksjoner. Videre fly carcass er motstandsdyktig mot standard homogenisering protokoller og krever en perle mill å sikre optimal metabolitten utvinning. Endelig, prøver analysert av GC-MS må gjennomgå kjemiske derivatization før oppdagelsen12. Mens tidligere publiserte metodene beskriver alle disse trinnene3,13,14, er en visuell protokoll som ville tillate nybegynneren reproduserbar generere høy kvalitetsdata fortsatt behov. Her viser vi hvordan å forberede Drosophila larver prøver for GC-baserte metabolomics analyse. Denne protokollen lar brukeren reproduserbar måle mange av små polar metabolitter som utgjør sentrale karbon metabolisme.

<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

Nylige fremskritt innen metabolomics har etablert frukt fly Drosophila melanogaster som en kraftig genetisk modell for å studere dyr metabolisme. Ved å kombinere stort utvalg av Drosophila genetisk verktøy muligheten til å kartlegge store swaths av mellomledd metabolisme, kan en metabolomics tilnærming åpenbare komplekse interaksjoner mellom kosthold, genotype, livshistorie hendelser og miljømessige signaler. I tillegg kan metabolomics studier oppdage romanen enzymatisk mekanismer og avdekke tidligere ukjente forbindelser mellom tilsynelatende ulike metabolske veier. For å lette mer utbredt bruk av denne teknologien blant Drosophila samfunnet, her vi gir en detaljert protokoll som beskriver hvordan å forberede Drosophila larver prøver gass kromatografi-massespektrometri (GC-MS)- basert metabolomic analyse. Våre protokollen inneholder beskrivelser av larver prøvetaking, metabolitten utvinning, kjemiske derivatization og GC-MS analyse. Vellykket gjennomføring av denne protokollen tillater brukere å måle den relative overfloden av små polar metabolitter, inkludert aminosyrer, sukker og organiske syrer involvert i Glykolysen og TCA sykluser.

Laktat dehydrogenase (dLDH) mutanter, som mangler dLDH aktivitet4og genetisk-matchet kontroller ble samlet Larvene midt-L2 og bearbeidet etter protokoll beskrevet ovenfor. Sammenlignet med kontrollene, utstilling mutant Larvene betydelige endringer i laktat, pyruvate og L-2-hydroxyglutarate4. Spectra anskaffet med Agilent GC6890-5973i MS system. Et eksempel på GC-MS spectra generere med våre protokollen er vist i <strong class="xf...

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 protokollen beskriver hvordan å forberede Drosophila Larvene GC-baserte metabolomic analyse.

Utarbeidelse av Drosophila larver prøver for gass kromatografi-massespektrometri (GC-MS)-basert 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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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-Cell Imaging av Drosophila melanogaster Tredje Instar Larval Brains

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 

Disseksjon av Drosophila melanogaster Tredje instar Larval Brain for Live-Cell Imaging