Name: gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil
Uploaded: 2012-05-19T14:00:00
Description: Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

यह काम डो ग्रेट लेक्स Bioenergy अनुसंधान केंद्र (डे - FC02 07ER64494 विज्ञान डो हिट कार्यालय) से अनुदान द्वारा समर्थित किया गया. हम डॉ. Xudong जांग और तकनीकी सहायक और प्रोटोकॉल को अंतिम रूप देने पर चर्चा अमूल्य टिप्पणी के लिए अपने समूह के सदस्यों के लिए आभारी हैं.

1. नमूना तैयार करना और एसिड निकालना <ol><li> क्षेत्र संग्रह के बाद रुक सूखी मिट्टी के नमूने. </li><li> पीस और एक गेंद मिल, मिट्टी चक्की, या एक मोर्टार और मूसल का उपयोग कर मिट्टी के नमूने homogenize. </li><li> एक 25 एमएल hydrolysis के कुप्प...

<ol>
	<li>Zelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+patterns+of+phospholipids+and+lipopolysaccharides+in+the+characterisation+of+microbial+communities+in+soil:+a+review.">Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review.</a> Biology and Fertility of Soils. 29, 111-129 (1999).</li><li>Kirk, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+studying+soil+microbial+diversity.">Methods of studying soil microbial diversity.</a> Journal of Microbiological Methods. 58, 169-188 (2004).</li><li>Joergensen, R. G., Emmerling, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+evaluating+human+impact+on+soil+microorganisms+based+on+their+activity,+biomass,+and+diversity+in+agricultural+soils.">Methods for evaluating human impact on soil microorganisms based on their activity, biomass, and diversity in agricultural soils.</a> Journal of Plant Nutrition and Soil Science. 169, 295-309 (2006).</li><li>Guggenberger, G., Frey, S. D., Six, J., Paustian, K., Elliott, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+and+fungal+cell-wall+residues+in+conventional+and+no-tillage+agroecosystems.">Bacterial and fungal cell-wall residues in conventional and no-tillage agroecosystems.</a> Soil Science Society of America Journal. 63, 1188-1198 (1999).</li><li>Amelung, W., Lal, R., Kimble, J. M., Follett, R. F., Stewart, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assessment Methods for Soil Carbon. , 233-270 (2001).</li><li>Guerrant, G. O., Moss, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+monosaccharides+as+aldononitrile,+O-methyloxime,+alditol,+and+cyclitol+acetate+derivatives+by+gas-chromatography.">Determination of monosaccharides as aldononitrile, O-methyloxime, alditol, and cyclitol acetate derivatives by gas-chromatography.</a> Analytical Chemistry. 56, 633-638 (1984).</li><li>Zhang, X., Amelung, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatographic+determination+of+muramic+acid,+glucosamine,+mannosamine,+and+galactosamine+in+soils.">Gas chromatographic determination of muramic acid, glucosamine, mannosamine, and galactosamine in soils.</a> Soil Biology and Biochemistry. 28, 1201-1206 (1996).</li><li>Liang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+molecular+ion+structure+for+aldononitrile+acetate+derivatized+muramic+acid.">Investigation of the molecular ion structure for aldononitrile acetate derivatized muramic acid.</a> Journal of Microbiological Methods. 86, 224-230 (2011).</li><li>He, H., Xie, H., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+GC/MS+technique+to+assess+15N+and+13C+incorporation+into+soil+amino+sugars.">A novel GC/MS technique to assess 15N and 13C incorporation into soil amino sugars.</a> Soil Biology and Biochemistry. 38, 1083-1091 (2006).</li><li>Glaser, B., Gross, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound-specific+delta+13C+analysis+of+individual+amino+sugars+-+a+tool+to+quantify+timing+and+amount+of+soil+microbial+residue+stabilization.">Compound-specific delta 13C analysis of individual amino sugars - a tool to quantify timing and amount of soil microbial residue stabilization.</a> Rapid Communications in Mass Spectrometry. 19, 1409-1416 (2005).</li><li>Liang, C., Balser, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+characterization+of+amino+sugar+aldononitrile+acetate+derivatives+used+for+isotope+enrichment+assessment+of+microbial+residues.">Mass spectrometric characterization of amino sugar aldononitrile acetate derivatives used for isotope enrichment assessment of microbial residues.</a> Soil Biology and Biochemistry. 42, 904-909 (2010).</li><li>Liang, C., Pedersen, J. A., Balser, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycoside+antibiotics+may+interfere+with+microbial+amino+sugar+analysis.">Aminoglycoside antibiotics may interfere with microbial amino sugar analysis.</a> Journal of Chromatography A. 1216, 5296-5301 (2009).</li></ol>

aldononitrile एसीटेट derivatized glucosamine और muramic एसिड के विश्लेषण के लिए प्रस्तुत जीसी आधारित विधि एक अपेक्षाकृत तेजी से इन अमीनो शर्करा, मिट्टी से निकाले यों विधि प्रदान करता है. डेरिवेटिव रासायनिक स्थिर हैं, और एक विश्ले...

<table border="1"><tbody><tr><td> अभिकर्मक का नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> Muramic एसिड </td><td> सिग्मा Aldrich </td><td> M2503-25mg </td></tr><tr><td> डी हाइड्रोक्लोराइड (+) - glucosamine </td><td> सिग्मा Aldrich </td><td> G1514-100g </td></tr><tr><td> एन मिथाइल-D-glucamine </td><td> सिग्मा Aldrich </td><td> M2004-500G </td></tr><tr><td> Myo - inositol </td><td> फिशर साइंटिफिक </td><td> A307003G025 </td></tr><tr><td> मेथनॉल (सूखा) </td><td> Acros ऑर्गेनिक्स </td><td> AC326950010 </td></tr><tr><td> 4 dimethylamino - पिरिडीन </td><td> Acros ऑर्गेनिक्स </td><td> AC148270050 </td></tr><tr><td> एथिल एसीटेट </td><td> VWR इंटरनेशनल </td><td> BJGC100 - 4 </td></tr><tr><td> Hydroxlamine हाइड्रोक्लोराइड </td><td> फिशर साइंटिफिक </td><td> H330 - 100 </td></tr><tr><td> पिरिडीन </td><td> फिशर साइंटिफिक </td><td> P368-500 </td></tr><tr><td> एसिटिक एनहाइड्राइड </td> &lt;p&gt; फिशर साइंटिफिक </td><td> A10 - 100 </td></tr><tr><td> Dichloromethane (क्लोराइड) </td><td> फिशर साइंटिफिक </td><td> D37 - 500 </td></tr><tr><td> हेक्सेन </td><td> फिशर साइंटिफिक </td><td> H303 - 4 </td></tr><tr><td> हाइड्रोक्लोरिक एसिड (6M) </td><td> फिशर साइंटिफिक </td><td> S456 - 4 </td></tr><tr><td> Hydroxylamine हाइड्रोक्लोराइड -15 एन </td><td> चिह्न सेवाएं </td><td> IN5280 </td></tr><tr><td> एसिटिक एनहाइड्राइड -2 एच (डी सी 6 4 ओ 3) </td><td> Acros ऑर्गेनिक्स </td><td> AC174670050 </td></tr><tr><td> यू डी ग्लूकोज सी -13 </td><td> कैम्ब्रिज आइसोटोप प्रयोगशाला </td><td> CLM-1396-1 </td></tr><tr><td> का अमोनियम sufate -15 एन </td><td> कैम्ब्रिज आइसोटोप प्रयोगशाला </td><td> एनएलएम-713-1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> उपकरणों के नाम </td><td> कंपनी </td><td> टाइप </td></tr><tr><td> तेजी से VAP </td&gt; <td> LabConco </td><td> +७,९०,००२ </td></tr><tr><td> Vacum पंप </td><td> KNF Neuberger </td><td> डी - 79,112 </td></tr><tr><td> Hydrolysis कुप्पी </td><td> फिशर साइंटिफिक </td><td> 06 423A </td></tr><tr><td> Derivatization microvial </td><td> फिशर साइंटिफिक </td><td> 06-100E </td></tr><tr><td> जीसी </td><td> Hewlett-Packard </td><td> 6890 </td></tr><tr><td> एमएस </td><td> Hewlett-Packard </td><td> 5972 </td></tr><tr><td> नियंत्रण रेखा के ईएसआई-TOF-एमएस </td><td> Agilent है </td><td> एक Agilent के 1200 श्रृंखला HPLC प्रणाली में Agilent नियंत्रण रेखा / एमएसडी-TOF युग्मित </td></tr></tbody></table>

gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

हम glucosamine और muramic मिट्टी से निकाले एसिड के aldonitrile एसीटेट डेरिवेटिव के जीसी आधारित विश्लेषण के लिए एक विधि प्रोटोकॉल का वर्णन. रासायनिक तंत्र की व्याख्या के लिए, हम भी एक व्युत्पन्न और आयन इलेक्ट्रॉन ionization पर गठित टुकड़े की संरचना की पुष्टि की रणनीति प्रस्तुत करते हैं.

मिट्टी में माइक्रोबियल अवशेष निर्धारण के लिए जीसी आधारित Aldononitrile एसीटेट derivatized Glucosamine और Muramic एसिड की जांच

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

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