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

Это исследование было поддержано частично грантов-в-помощь по научным исследованиям для сложных поисковых исследований (JK), а также научных исследований (A) (JK и SM) от Министерства Образования, Культуры, Спорта, Науки и Технологии Японии . RIKEN FPR стипендий (RCE), при условии дополнительной поддержки. Авторы выражают благодарность д-ра. Eisuke Chikayama, Yasuyo Sekiyama и Мами Окамото для оказания технической помощи с помощью ЯМР и статистического анализа.

1. Подготовка фильтров для удаления экстрагируемых <ol><li> Использование 25-мм диаметром 0,22 мкм поры размером Durapore PVDF гидрофильные фильтры (Millipore). Место фильтры в чистом стакане 500 мл Pyrex с помощью пинцета. Предварительно промыть три раза дистиллированной водой. Swirl хорошо, как вы промыть, ч...

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Метод фильтрации и метаболитов добычи продемонстрировали здесь позволяет микробной биомассы планктонных должны быть собраны в достаточном количестве для метаболомики ЯМР. В то время как только добыча водно-растворимых метаболитов на основе KPI и 1D 1 H ЯМР показал, другие раствори...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу </td><td> Комментарии </td></tr><tr><td> 0,22 мкм гидрофильный Durapore PVDF фильтров, 25 мм </td><td> Millipore </td><td> GVWP02500 </td><td></td></tr><tr><td> Микроанализ держатель фильтра 25 мм, оплавленных стеклянных поддержки </td><td> Millipore </td><td> XX1002500 </td><td></td></tr><tr><td> 3-место многообразии, 47 мм, нержавеющая сталь </td><td> Millipore </td><td> XX2504735 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td> Вако </td><td> 169-04245 </td><td></td></tr><tr><td> K 2 HPO 4 </td><td> Вако </td><td> 164-04295 </td><td></td></tr><tr><td> Оксид дейтерия 2 H&gt; 90% </td><td> Campridge Изотопный 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-АМ4 </td><td> Из нержавеющей стали входят дробилки </td></tr><tr><td> Thermomixer комфорт </td><td> Эппендорф </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> Вакуумный испаритель </td><td> EYELA </td><td> CVE-3100 </td><td></td></tr><tr><td> ЯМР </td><td> Bruker </td><td> DRX-500 с 5 мм TXI зонд </td><td></td></tr><tr><td> Спектральный биннинга инструмент </td><td> Первоначально разработанный </td><td> FT2DB </td><td> <a href="https://database.riken.jp/ecomics/">https://database.riken.jp/ecomics/</a> </td></tr><tr><td> Метаболитов инструмент аннотаций и баз данных </td><td> Первоначально разработанный </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 is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

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

Метод метаболитов извлечения из микробных сообществ планктона представлена. Всего выборки сообщества достигается путем фильтрации на специально подготовленных фильтров. После лиофилизации, водно-растворимых метаболитов выделяются. Такой подход позволяет для применения экологических метаболомики к транс-omics исследования природных и экспериментальных микробных сообществ.

Концентрация метаболита с низкой плотностью планктонных сообществ экологической метаболомики использованием ядерного магнитного резонанса

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

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