चयापचयों की परमाणु चुंबकीय अनुनाद स्पेक्ट्रोस्कोपी का उपयोग पर्यावरण Metabolomics के लिए कम घनत्व planktonic समुदाय से एकाग्रता
Summary
माइक्रोबियल planktonic समुदायों से metabolite का निष्कर्षण के लिए एक विधि प्रस्तुत किया है. पूरे समुदाय के नमूने निस्पंदन द्वारा विशेष रूप से तैयार फिल्टर पर हासिल की है. Lyophilization के बाद, जलीय घुलनशील चयापचयों निकाले जाते हैं. इस दृष्टिकोण से पर्यावरण metabolomics की प्राकृतिक या प्रयोगात्मक सूक्ष्म समुदायों के पार omics जांच करने के लिए आवेदन के लिए अनुमति देता है.
Abstract
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–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.
Protocol
Discussion
निस्पंदन और metabolite निष्कर्षण विधि यहाँ का प्रदर्शन माइक्रोबियल planktonic बायोमास के लिए पर्याप्त राशि के में एनएमआर metabolomics के लिए एकत्र होने की अनुमति देता है. जबकि जलीय घुलनशील चयापचयों का उपयोग कर KPI और 1D 1 ?…
Declarações
The authors have nothing to disclose.
Acknowledgements
इस शोध में भाग द्वारा समर्थित किया गया अनुदान सहायता में शिक्षा, संस्कृति, खेल, विज्ञान मंत्रालय से खोजपूर्ण (जे), अनुसंधान और वैज्ञानिक अनुसंधान (ए) (जे और एस) को चुनौती देने के लिए वैज्ञानिक अनुसंधान के लिए, और प्रौद्योगिकी, जापान . एक आरआईकेईएन fpr फैलोशिप (RCE) अतिरिक्त सहायता प्रदान की है. लेखकों डीआरएस के प्रति अपनी कृतज्ञता व्यक्त करते हैं. Eisuke Chikayama, Yasuyo Sekiyama और एनएमआर और सांख्यिकीय विश्लेषण के साथ तकनीकी सहायता के लिए ममी Okamoto.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| 0.22 μm hydrophilic Durapore PVDF filters, 25 mm | Millipore | GVWP02500 | |
| Microanalysis Filter Holder, 25 mm, fritted glass support | Millipore | XX1002500 | |
| 3-place manifold, 47 mm, stainless steel | Millipore | XX2504735 | |
| KH2PO4 | Wako | 169-04245 | |
| K2HPO4 | Wako | 164-04295 | |
| Deuterium oxide, 2H > 90% | Campridge Isotope Laboratoties | DLM-4 | |
| DSS | Fluka | 92754 | |
| Automill | Tokken | TK-AM4 | Stainless steel crushers included |
| Thermomixer comfort | Eppendorf | 5355 000.011 | |
| Bioruptor | Diagenode | UCD-200 | |
| Vacuum evaporator | EYELA | CVE-3100 | |
| NMR | Bruker | DRX-500 with 5 mm-TXI probe | |
| Spectral binning tool | Originally developed | FT2DB | https://database.riken.jp/ecomics/ |
| Metabolite annotation tool and database | Originally developed | SpinAssign | http://prime.psc.riken.jp/?action=nmr_search |
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