Name: concentration of metabolites from low-density planktonic communities for environmental metabolomics using nuclear magnetic resonance spectroscopy
Uploaded: 2012-04-07T14:00:00
Description: Watch this Scientific Journal Video about Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Denne forskning blev støttet delvist af tilskud-in-Støtte til videnskabelig forskning for at anfægte sonderende forskning (JK), og videnskabelig forskning (A) (JK og SM) fra Ministeriet for Undervisning, Kultur, Sport, Videnskab og Teknologi, Japan . En Riken FPR fællesskab (RCE) gav yderligere støtte. Forfatterne udtrykker deres taknemmelighed til Drs. Eisuke Chikayama, Yasuyo Sekiyama og Mami Okamoto til teknisk bistand med NMR og statistiske analyser.

1. Filter Forberedelse til Fjern ekstraherbare <ol><li> Anvende 25 mm diameter 0,22 um porestørrelse Durapore PVDF hydrofile filtre (Millipore). Placer filtre i et rent 500 ml Pyrex bægerglas med en pincet. Forskylning tre gange med destilleret vand. Hvirvel og man skylle at forhindre filtrene i at klæbe til hinanden. Tilsæt 300 ml Milli-Q (Millipore) eller tilsvarende vand af høj kvalitet. Autoklaveres for at lette fuldstændig fjernelse af ekstraherbare fra filtrene. </li><li> Hæld Milli-Q og igen tre gange sk...

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Den filtrering og metabolit ekstraktion metode demonstreret her giver mulighed for mikrobiel planktoniske biomasse, der skal indsamles i tilstrækkelig mængde til NMR metabolomics. Mens kun ekstraktion af vandopløselige metabolitter med KPi og 1D 1H-NMR er vist, kan andre ekstraktionsmidler og spektroskopiske fremgangsmåder anvendes. Et godt eksempel er anvendelsen af deutereret methanol som en semi-polært opløsningsmiddel, som har vist sig at frembringe overlegen NMR-spektre fra heterogene prøver og er...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> 0,22 um hydrofile Durapore PVDF-filtre, 25 mm </td><td> Millipore </td><td> GVWP02500 </td><td></td></tr><tr><td> Mikroanalysen Filter Holder, 25 mm, glasfilter støtte </td><td> Millipore </td><td> XX1002500 </td><td></td></tr><tr><td> 3-sted manifold, 47 mm, rustfrit stål </td><td> Millipore </td><td> XX2504735 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Wako </td><td> 169-04245 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> Wako </td><td> 164-04295 </td><td></td></tr><tr><td> Deuteriumoxid, 2 H&gt; 90% </td><td> Campridge Isotope Laboratoties </td><td> DLM-4 </td><td></td<html> &gt; </tr><tr><td> DSS </td><td> Fluka </td><td> 92754 </td><td></td></tr><tr><td> Automill </td><td> Tokken </td><td> TK-AM4 </td><td> Rustfrit stål knusere medfølger </td></tr><tr><td> Thermomixer komfort </td><td> Eppendorf </td><td> 5355 000.011 </td><td></td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 </td><td></td></tr><tr><td> Vakuuminddamper </td><td> EYELA </td><td> CVE-3100 </td><td></td></tr><tr><td> NMR </td><td> Bruker </td><td> DRX-500 med 5 mm TXI probe </td><td></td></tr><tr><td> Spektral Binning værktøj </td><td> Oprindeligt udviklet </td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td> Metabolit annotation værktøj og database </td><td> Oprindeligt udviklet </td><td> SpinAssign </td><td>= Nmr_search &quot;&gt; http://prime.psc.riken.jp/?action=nmr_search </td></tr></tbody></table>

concentration of metabolites from low-density planktonic communities for environmental metabolomics using nuclear magnetic resonance spectroscopy

Environmental metabolomics er en spirende felt, der fremmer ny forståelse for, hvordan organismer reagerer på og interagere med miljøet og hinanden på det biokemiske niveau 1. Kernemagnetisk resonans (NMR) spektroskopi er en af ​​flere teknikker, herunder gaskromatografi-massespektrometri (GC-MS), med betydelig lovende for sådanne undersøgelser. Fordele ved NMR er, at det er egnet til målrettede analyser, giver strukturel information og spektre kan forespørges i kvantitative og statistiske manerer mod nyligt tilgængelige databaser i de enkelte metabolit spektre 2,3. Desuden kan NMR-spektrale data kombineres med data fra andre omik niveauer (f.eks transcriptomics, genomforskning) til at give en mere omfattende forståelse af de fysiologiske reaktioner i taxa til hinanden og miljøet 4,5,6. Imidlertid NMR er mindre følsomme end andre metabolom teknikker, hvilket gør det vanskeligt at aplag af naturlige mikrobielle systemer, hvor prøvepopulationer kan være med lav massefylde og metabolitkoncentrationer lav sammenlignet med metabolitter fra veldefinerede og udtrækkes kilder såsom hele væv Biofluids eller celle-kulturer. Følgelig har de få direkte miljømæssige metabolom undersøgelser af mikrober udført til dato været begrænset til kultur-baserede eller let defineret høj densitet økosystemer, såsom vært-symbiont systemer, konstrueret co-kulturer eller manipulationer af tarmen miljø, hvor en stabil isotop mærkning kan være yderligere anvendes til at forøge NMR-signaler 7,8,9,10,11,12. Metoder, som letter koncentrationen og indsamling af miljøprøver metabolitter i koncentrationer der er egnede til NMR mangler. Siden seneste opmærksomhed er blevet givet til de miljømæssige metabolomics af organismer i vandmiljøet, hvor meget af den energi og materiale flow er medieret af planktoniske samfund 13,14, har vi udviklet en metode til koncentrationning og udvinding af hele samfundet metabolitter fra planktoniske mikrobielle systemer ved filtrering. Kommercielt tilgængelige hydrofile poly-1 ,1-difluoroethene (PVDF) filtre specielt behandlet for fuldstændigt at fjerne ekstraherbare, som ellers kan vises som forureninger i de efterfølgende analyser. Disse behandlede filtre anvendes derefter til at filtrere miljømæssige eller eksperimentelle prøver af interesse. Filtre indeholdende det våde prøvematerialet er lyofiliseret og vandopløselige metabolitter ekstraheres direkte ved konventionel NMR-spektroskopi ved anvendelse af en standardiseret kaliumphosphatpuffer ekstraktionsbuffer 2. Data fra disse metoder kan analyseres statistisk for at identificere meningsfulde mønstre, eller integreret med andre omik niveauer for forståelse af samfund og økosystem funktion.

Watch this Scientific Journal Video about Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy at JoVE.com

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

En metode til metabolit ekstraktion fra mikrobielle planktoniske samfund præsenteres. Hele samfund prøvetagning opnås ved filtrering på specielt forberedte filtre. Efter lyofilisering, er vandopløselige metabolitter ekstraheres. Denne fremgangsmåde giver mulighed for anvendelse af miljømæssige metabolomics til trans-omik undersøgelser af naturlige eller eksperimentel mikrobielle samfund.

Koncentration af metabolitter fra low-density Plankton Fællesskaber for miljøet Metabolomics med kernemagnetisk resonans-spektroskopi

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