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

Dette arbejde blev støttet af tilskud fra DOE Great Lakes i Bioenergi Research Center (DOE BER Office of Science DE-FC02-07ER64494). Vi er taknemmelige for Dr. Xudong Zhang og hans gruppe medlemmer til nyttige tekniske drøftelser og uvurderlige Bemærkninger til færdiggørelsen af ​​protokollen.

1. Prøveforberedelse og syreekstraktion <ol><li> Frysetørre jordprøver efter feltet kollektion. </li><li> Male og homogeniseres jordprøver anvendelse af en kuglemølle, jord kværn eller en morter og pistil. </li><li> Afvej jordprøver (indeholdende&gt; 0,3 mg N) i en 25 ml hydrolyse kolbe. </li><li> Tilsæt 10 ml 6M HCI i hver hydrolyse kolbe fyldes N2-gas i kolberne, og låg. </li><li> Hydrolysere ved 105 ° C i en inkubator i 8 timer under anvendelse af en auto kontaktur. </li></ol><p class="jove_tit...

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	<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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Den fremlagte GC-metode til analyse af aldononitrile acetat derivatiserede glucosamin og muramic syre giver en relativt hurtig metode til at kvantificere disse aminosukkere, udvundet af jord. De derivater er kemisk stabile, og kan bestemmes i en analyse. Metoden er ikke begrænset til jordprøver og kan forenkles for prøver fra ikke-jord matrix. Vakuumpumpen anvendes i denne fremgangsmåde er designet til at være modstandsdygtig over for syre. Vi foreslog endvidere, at oprette en base fæl...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Muramic syre </td><td> Sigma-Aldrich </td><td> M2503-25mg </td></tr><tr><td> D-(+)-glucosamin-hydrochlorid </td><td> Sigma-Aldrich </td><td> G1514-100G </td></tr><tr><td> N-methyl-D-glucamin </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 (tør) </td><td> Acros Organics </td><td> AC326950010 </td></tr><tr><td> 4-dimethylamino-pyridin </td><td> Acros Organics </td><td> AC148270050 </td></tr><tr><td> Ethylacetat </td><td> VWR International </td><td> BJGC100-4 </td></tr><tr><td> Hydroxlamine hydrochlorid </td><td> Fisher Scientific </td><td> H330-100 </td></tr><tr><td> Pyridin </td><td> Fisher Scientific </td><td> P368-500 </td></tr><tr><td> Eddikesyreanhydrid </td> &lt;td&gt; Fisher Scientific </td><td> A10-100 </td></tr><tr><td> Dichlormethan (methylenchlorid) </td><td> Fisher Scientific </td><td> D37-500 </td></tr><tr><td> Hexan </td><td> Fisher Scientific </td><td> H303-4 </td></tr><tr><td> Saltsyre (6 M) </td><td> Fisher Scientific </td><td> S456-4 </td></tr><tr><td> Hydroxylaminhydrochlorid -15 N </td><td> Ikon tjenester </td><td> IN5280 </td></tr><tr><td> Eddikesyreanhydrid -2 H (D6 C4 O 3) </td><td> Acros Organics </td><td> AC174670050 </td></tr><tr><td> D-glucose-U -13 C </td><td> Cambridge isotop laboratoriet </td><td> CLM-1396-1 </td></tr><tr><td> Ammonium sufate -15 N </td><td> Cambridge isotop laboratoriet </td><td> NLM-713-1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Navn udstyr </td><td> Firma </td><td> Type </td></tr><tr><td> Rapid-Vap </td&gt; <td> Labconco </td><td> 790002 </td></tr><tr><td> Vakuum, hvorved pumpe </td><td> KNF Neuberger </td><td> D-79.112 </td></tr><tr><td> Hydrolyse kolbe </td><td> Fisher Scientific </td><td> 06 423A </td></tr><tr><td> Derivatisering eppendorfrøret </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> En Agilent 1200 udgaven af ​​HPLC system koblet til en 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

Kvantitative metoder til at karakterisere mikroorganismer er afgørende for en bredere forståelse af den mikrobielle status og funktion i økosystemerne. Aktuelle strategier for mikrobiel analyse omfatter både traditionelle laboratorie kultur-afhængige teknikker og de ​​er baseret på direkte udvinding og bestemmelse af visse biomarkører 1, 2. Få blandt mangfoldigheden af ​​mikrobielle arter bebo jorden kan dyrkes, så kultur-afhængige metoder medføre store afvigelser, en begrænsning fraværende i biomarkør analyse. Den glucosamin, mannosamin, galactosamin og muramic syre er blevet godt tjent som foranstaltninger af både levende og døde mikrobiel masse af disse glucosamin (hyppigst forekommende) og muramic syre (unik fra bakteriecellen) er de vigtigste bestanddele i jorden systemerne 3 , 4. Men manglende viden om analysen begrænser den brede popularization mellem videnskabelige jævnaldrende. Blandt alle existikke analysemetoder, derivatisering til aldononitrile acetater efterfulgt af GC-analyse har vist sig som en god mulighed for med hensyn til optimalt afbalancere præcision, følsomhed, enkelhed, god kromatografisk separation, og stabilitet ved prøveopbevaringsperioden 5. Her præsenterer vi en detaljeret protokol for en pålidelig og relativt enkel analyse af glucosamin og muramic syre fra jorden efter deres konvertering til aldononitrile acetater. Protokollen hovedsageligt består af fire trin: syre fordøjelse, prøve rensning, derivatisering og GC beslutsomhed. Den trin-for-trin procedure er modificeret i henhold til tidligere publikationer 6, 7. Desuden præsenterer vi en strategi for strukturelt validere molekylære ion af derivatet og dets ion-fragmenter dannet ved elektron ionisering. Vi anvendte GC-EI-MS-SIM, LC-ESI-TOF-MS og isotopisk mærkede reagenser til bestemmelse af molekylvægten af ​​aldononitrile acetat derivatiseret glucosamin og muramidsyre, anvendte vi massen forskydning af isotop-mærkede derivater af ion-spektret til at undersøge ion fragmenter af hver derivater 8. Ud over den teoretiske belysningen vil validering af molekylære ion af derivatet og ion-fragmenter være nyttigt at forskere ved hjælp δ 13C eller ion fragmenter af disse biomarkører i biogeokemiske undersøgelser 9, 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

Beskriver vi en fremgangsmåde protokol for GC-analyse af aldonitrile acetat derivater af glucosamin og muramic syre ekstraheret fra jord. Til belysning af den kemiske mekanisme også præsenteres en strategi for at bekræfte strukturen af ​​derivatet og ion-fragmenterne dannet ved elektron ionisering.

GC-baseret detektion af Aldononitrile Acetat Derivatiseret Glucosamin og Muramic Acid for Mikrobiel Rest bestemmelse i jord

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