This work was supported by grants from DOE Great Lakes Bioenergy Research Center (DOE BER office of Science DE-FC02-07ER64494). We are grateful to Dr. Xudong Zhang and his group members for helpful technical discussions and invaluable comments on finalizing the protocol.

1. Sample Preparation and Acid Extraction
<ol>
	<li>Freeze-dry soil samples after field collection.</li>
	<li>Grind and homogenize soil samples using a ball mill, soil grinder, or a mortar and pestle.</li>
	<li>Weigh soil samples (containing &gt; 0.3 mg N) into a 25 mL hydrolysis flask.</li>
	<li>Add 10 mL 6M HCl into each hydrolysis flask, fill N2 gas in the flasks, and cap tightly.</li>
	<li>Hydrolyze at 105 &deg;C in an incubator for 8 hours using an auto timer switch.</li>
</ol>
2. ...

<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. 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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. 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The presented GC-based method for the analysis of aldononitrile acetate derivatized glucosamine and muramic acid provides a relatively rapid method to quantify these amino sugars, extracted from soil. The derivatives are chemically stable, and can be determined in one analysis. The method is not restricted to soil samples, and can be simplified for samples from non-soil matrix.
The vacuum pump used in this method is built to be resistant to acid. We further suggest setting up a base trap to p...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Muramic acid</td>
 <td>Sigma-Aldrich</td>
 <td>M2503-25MG</td>
 </tr>
 <tr>
 <td>D-(+)-glucosamine hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>G1514-100G</td>
 </tr>
 <tr>
 <td>N-methyl-D-glucamine</td>
 <td>Sigma-Aldrich</td>
 <td>M2004-500G</td>
 </tr>
 <tr>
 <td>Myo-inositol</td>
 <td>Fisher Scientific</td>
 <td>A307003G025</td>
 </tr>
 <tr>
 <td>Methanol (dry)</td>
 <td>Acros organics</td>
 <td>AC326950010</td>
 </tr>
 <tr>
 <td>4-dimethylamino-pyridine</td>
 <td>Acros organics</td>
 <td>AC148270050</td>
 </tr>
 <tr>
 <td>Ethyl acetate</td>
 <td>VWR International</td>
 <td>BJGC100-4</td>
 </tr>
 <tr>
 <td>Hydroxlamine hydrochloride </td>
 <td>Fisher Scientific</td>
 <td>H330-100</td>
 </tr>
 <tr>
 <td>Pyridine</td>
 <td>Fisher Scientific</td>
 <td>P368-500</td>
 </tr>
 <tr>
 <td>Acetic anhydride</td>
 <td>Fisher Scientific</td>
 <td>A10-100</td>
 </tr>
 <tr>
 <td>Dichloromethane (Methylene chloride)</td>
 <td>Fisher Scientific</td>
 <td>D37-500</td>
 </tr>
 <tr>
 <td>Hexane</td>
 <td>Fisher Scientific</td>
 <td>H303-4</td>
 </tr>
 <tr>
 <td>Hydrochloric acid (6M)</td>
 <td>Fisher Scientific</td>
 <td>S456-4</td>
 </tr>
 <tr>
 <td>Hydroxylamine hydrochloride-15N</td>
 <td>Icon services</td>
 <td>IN5280</td>
 </tr>
 <tr>
 <td>Acetic anhydride-2H (D6C4O3)</td>
 <td>Acros organics</td>
 <td>AC174670050</td>
 </tr>
 <tr>
 <td>D-glucose-U-13C</td>
 <td>Cambridge isotope lab</td>
 <td>CLM-1396-1</td>
 </tr>
 <tr>
 <td>Ammonium sufate-15N </td>
 <td>Cambridge isotope lab</td>
 <td>NLM-713-1</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Name of the equipment</td>
 <td>Company</td>
 <td>Type</td>
 </tr>
 <tr>
 <td>Rapid-Vap</td>
 <td>LabConco</td>
 <td>790002 </td>
 </tr>
 <tr>
 <td>Vacum pump</td>
 <td>KNF Neuberger</td>
 <td>D-79112</td>
 </tr>
 <tr>
 <td>Hydrolysis flask</td>
 <td>Fisher Scientific </td>
 <td>06 423A</td>
 </tr>
 <tr>
 <td>Derivatization microvial</td>
 <td>Fisher Scientific</td>
 <td>06-100E</td>
 </tr>
 <tr>
 <td>GC</td>
 <td>Hewlett-Packard</td>
 <td>6890</td>
 </tr>
 <tr>
 <td>MS</td>
 <td>Hewlett-Packard</td>
 <td>5972</td>
 </tr>
 <tr>
 <td>LC-ESI-TOF-MS</td>
 <td>Agilent</td>
 <td>An Agilent 1200 series HPLC system coupled to an Agilent LC/MSD-TOF </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

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

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

gc-based-detection-aldononitrile-acetate-derivatized-glucosamine

Research

JoVE Journal

Biology

16.7K visualizações.  University of Wisconsin, Madison. Nós descrevemos um método para o protocolo de análise de GC-Based dos derivados aldonitrile acetato de glucosamina e ácido murâmico extraído do solo. Para elucidação do mecanismo química, também apresentar uma estratégia para confirmar a estrutura do derivado e os fragmentos de iões formados mediante ionização de electrões.