代谢物的浓度从低密度浮游社区的环境代谢，利用核磁共振光谱
Summary
从微生物浮游社区的代谢产物提取方法。抽样实现整个社会上特意准备的过滤器过滤。冷冻干燥后，溶于水，代谢产物中提取。这种方法用于环境代谢的自然或实验的微生物群落的跨组学研究中的应用。
Abstract
环境代谢组学是一个新兴的领域有了新的认识，促进生物体是如何应对和交互环境和对方在生化水平1。核磁共振（NMR）光谱是多种技术，包括气相色谱 – 质谱（GC-MS法）之一，此类研究大有前途。核磁共振的优点是，它是适用于不相关的分析，提供结构化信息和光谱可以对最近2,3个人代谢物谱数据库查询数量和统计的举止。此外，核磁共振光谱数据可与其他组学水平（如转录，基因组学）的数据，以提供更全面的了解类群的生理反应，彼此和环境4,5,6。然而，核磁共振是比其他代谢组学技术的敏感，使其难以AP铺设天然微生物系统，样本人群低密度及代谢物浓度比较低的代谢物的定义和随时提取来源，如整个组织，biofluids或细胞培养。因此，一些直接环境日期进行微生物的代谢物的研究一直局限于文化为基础的或容易定义的高密度的生态系统，如主机的共生系统，构建联合培养或肠道内环境的稳定同位素标记可以操作此外用于增强核磁共振信号7,8,9,10,11,12。核磁共振适合浓度的方法，促进环境代谢物的浓度和收集所缺乏的。由于近期关注已在水生环境，其中大部分能量和物质流介导由的浮游社区13,14生物体对环境的代谢，我们已经开发出一种浓度的方法TION和全社会的代谢产物从浮游微生物系统过滤提取。市售亲水性的聚-1 ,1-difluoroethene（PVDF）过滤器是经过特殊处理，以彻底清除萃取物，可作为后续分析中的污染物，否则会出现。然后，这些治疗的过滤器用于过滤利益的环境或实验样品。湿样品材料的过滤器含有冻干和使用一个标准化的钾磷酸盐提取液常规核磁共振光谱直接溶于水的代谢产物中提取。可以从这些方法得出的数据进行统计分析，找出有意义的模式，或集成水平与其他组学为社会和生态系统功能的全面了解。
Protocol
1。过滤删除萃取物的制备使用25毫米直径0.22微米孔径Durapore PVDF（Millipore公司）亲水性过滤器。放置在一个干净的500毫升耐热烧杯过滤器，使用镊子。前用蒸馏水冲洗三次。漩涡以及你冲洗过滤器，以防止从坚持彼此。加入300毫升的Milli-Q（Millipore公司）或相当于高品质的水。釜萃取物彻底清除，以方便从过滤器。 倒掉的Milli-Q和再次三重冲洗过滤器，这个时候用Milli-Q。使用镊子个?…
Discussion
这里展示的过滤和代谢产物提取方法允许在收集足够数量的核磁共振代谢微生物浮游生物。虽然只溶于水的使用KPI和1D 1 H核磁共振的代谢产物提取证明，其他的提取溶剂和光谱方法可以使用。一个有用的例子是使用氘代甲醇作为一个半极性溶剂，这已被证明产生优越的核磁共振光谱，从异构样品及顺磁离子污染不敏感，在15个海洋样品发现。在这种情况下，从上面提取的颗粒应保?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了部分赠款援助具有挑战性的探索性研究（JK），科研（一）（JK和SM），从教育，文化，体育，科学部，科研和技术，日本。一个RIKEN的玻璃钢奖学金“（RCE），提供额外的支持。作者表达自己的谢意博士。英介Chikayama Yasuyo Sekiyama和冈本真身用核磁共振和统计分析的技术援助。
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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Tags
ConcentrationMetabolitesLow-densityPlanktonic CommunitiesEnvironmental MetabolomicsNuclear Magnetic Resonance SpectroscopyNMR SpectroscopyGas Chromatography-mass SpectrometryGC-MSUntargeted AnalysesStructural InformationQuantitative AnalysisStatistical AnalysisMetabolite Spectra DatabasesOmics LevelsTranscriptomicsGenomicsPhysiological ResponsesTaxaSensitivityNatural Microbial Systems
Cite This Article
Everroad, R. C., Yoshida, S., Tsuboi, Y., Date, Y., Kikuchi, J., Moriya, S. Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy. J. Vis. Exp. (62), e3163, doi:10.3791/3163 (2012).