Name: using capillary electrophoresis to quantify organic acids from plant tissue: a test case examining coffea arabica seeds
Uploaded: 2016-11-12T14:00:00
Description: Watch this Scientific Journal Video about Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds at JoVE.com

The authors would like to acknowledge the financial support of this project by The J.M. Smucker company.

1. Probenvorbereitung <ol><li> Montieren Sie Proben für die kurzkettigen Carbonsäure (SCCA) Extraktion. Bereiten 1,0 g Kaffee Saatguts zu einer Zeit, die ausreichend Probe sicherzustellen wird nach der Verarbeitung verbleiben. <ol><li> Wenn Proben vor dem Schleifprozess eingefroren wurden, halten Sie die während der Verarbeitung gefroren Gewebe Frost / Tau-Schäden und Probe Oxidation zu verhindern. Entfernen Sie die Probe aus dem Kühl- oder unter Null Lagerung nur zum Schleifen erforderlich. </li><li> Blitzeis fr...

<ol>
	<li>Ara&#250;jo, W. L., Nunes-Nesi, A., Nikoloski, Z., Sweetlove, L. J., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Control+and+Regulation+of+the+Tricarboxylic+Acid+Cycle+in+Photosynthetic+and+Heterotrophic+Plant+Tissues:+TCA+Control+and+Regulation+in+Plant+Tissues.">Metabolic Control and Regulation of the Tricarboxylic Acid Cycle in Photosynthetic and Heterotrophic Plant Tissues: TCA Control and Regulation in Plant Tissues.</a> Plant Cell Environ. 35 (1), 1-21 (2012).</li><li>Finkemeier, I., Konig, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+Analysis+of+the+Role+of+Carboxylic+Acids+in+Metabolite+Signaling+in+Arabidopsis+Leaves.">Transcriptomic Analysis of the Role of Carboxylic Acids in Metabolite Signaling in Arabidopsis Leaves.</a> Plant Physiol. 162 (1), 239-253 (2013).</li><li>Doyle, M. P., Buchanan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food Microbiology: Fundamentals and Frontiers. , (2013).</li><li>T&#367;ma, P., Samcov&#225;, E., &#352;tul&#236;k, K. <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+the+Spectrum+of+Low+Molecular+Mass+Organic+Acids+in+Urine+by+Capillary+Electrophoresis+with+Contactless+Conductivity+and+Ultraviolet+Photometric+Detection-An+Efficient+Tool+for+Monitoring+of+Inborn+Metabolic+Disorders.">Determination of the Spectrum of Low Molecular Mass Organic Acids in Urine by Capillary Electrophoresis with Contactless Conductivity and Ultraviolet Photometric Detection-An Efficient Tool for Monitoring of Inborn Metabolic Disorders.</a> Anal Chim Acta. 685 (1), 84-90 (2011).</li><li>L&#243;pez-Bucio, J., Nieto-Jacobo, M. F., Ram&#305;&#769;rez-Rodr&#305;&#769;guez, V., Herrera-Estrella, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+Acid+Metabolism+in+Plants:+From+Adaptive+Physiology+to+Transgenic+Varieties+for+Cultivation+in+Extreme+Soils.">Organic Acid Metabolism in Plants: From Adaptive Physiology to Transgenic Varieties for Cultivation in Extreme Soils.</a> Plant Sci. 160 (1), 1-13 (2000).</li><li>Cebolla-Cornejo, J., Valc&#225;rcel, M., Herrero-Mart&#236;nez, J. M., Rosell&#242;, S., Nuez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Efficiency+Joint+CZE+Determination+of+Sugars+and+Acids+in+Vegetables+and+Fruits:+CE+and+CEC.">High Efficiency Joint CZE Determination of Sugars and Acids in Vegetables and Fruits: CE and CEC.</a> Electrophoresis. 33 (15), 2416-2423 (2012).</li><li>Rosello, S., Galiana-Balaguer, L., Herrero-Martinez, J. M., Maquieira, A., Nuez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+the+Main+Organic+Acids+and+Carbohydrates+Involved+in+Tomato+Flavour+Using+Capillary+Zone+Electrophoresis.">Simultaneous Quantification of the Main Organic Acids and Carbohydrates Involved in Tomato Flavour Using Capillary Zone Electrophoresis.</a> J Sci Food Agr. 82 (10), 1101-1106 (2002).</li><li>Wasielewska, M., Banel, A., Zygmunt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+in+Determination+of+Low+Molecular+Mass+Organic+Acids.">Capillary Electrophoresis in Determination of Low Molecular Mass Organic Acids.</a> Int J Environ Sci Dev. 5 (4), 417-425 (2014).</li><li>Galli, V., Garc&#236;a, A., Saavedra, L., Barbas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+for+Short-Chain+Organic+Acids+and+Inorganic+Anions+in+Different+Samples.">Capillary Electrophoresis for Short-Chain Organic Acids and Inorganic Anions in Different Samples.</a> Electrophoresis. 24 (1213), 1951-1981 (2003).</li><li>Klampfl, 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+Organic+Acids+by+CE+and+CEC+Methods.">Determination of Organic Acids by CE and CEC Methods.</a> Electrophoresis. 28 (19), 3362-3378 (2007).</li><li>Kenney, B. F. <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+Organic+Acids+in+Food+Samples+by+Capillary+Electrophoresis.">Determination of Organic Acids in Food Samples by Capillary Electrophoresis.</a> J Chromatogr A. 546, 423-430 (1991).</li><li>Galli, V., Barbas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+for+the+Analysis+of+Short-Chain+Organic+Acids+in+Coffee.">Capillary Electrophoresis for the Analysis of Short-Chain Organic Acids in Coffee.</a> J Chromatogr A. 1032 (1-2), 299-304 (2004).</li><li>Schmitt-Kopplin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis:+Methods+and+Protocols.">Capillary Electrophoresis: Methods and Protocols.</a> Methods in Molecular Biology. , 384 (2008).</li><li>Nollet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatographic analysis of the environment 3rd ed. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ElixerOA Organic Acids/Anions Operating and Instruction Manual, MicroSolv Technology Corperation. , (2001).</li><li>Dahlen, J., Hagberg, J., Karlsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+low+molecular+weight+organic+acids+in+water+with+capillary+zone+electrophoresis+employing+indirect+photometric+detection.">Analysis of low molecular weight organic acids in water with capillary zone electrophoresis employing indirect photometric detection.</a> Fresenius J. Anal. Chem. 366 (5), 488-493 (2000).</li><li>Ibanez, A. B., Bauer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+method+for+the+determination+of+organic+acids+in+dilute+acid+pretreated+biomass+hydrolysate+by+liquid+chromatography-time-of-flight+mass+spectroscopy.">Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectroscopy.</a> Biotech. For Biofuels. 7 (145), (2014).</li></ol>

Wie bei jeder analytischen Technik gibt es mehrere kritische Faktoren, die die Qualität und die Zuverlässigkeit der erzeugten Daten erheblich auswirken können. Erstens ist es wichtig Proben effizient zu verarbeiten, mit einem Minimum von Gefrier- / Tau-Zyklen. Wiederholtes Einfrieren und Auftauen können die chemische Zusammensetzung der Probe vor der Verarbeitung oder Analyse beeinträchtigen. Zweitens ist es wichtig, die Schritte des Protokolls für alle Proben konstant und gleichmäßig aufzubringen. Technische Fe...

Carboxylic acids are organic compounds containing one or more terminal carboxyl functional groups, each attached to an R-group containing one or more carbons (R-C[O]OH). Short chain, low molecular weight carboxylic acids (short chain carboxylic acids, SCCAs) containing between one and six carbons, are essential components of cellular respiration, and function in several biochemical pathways necessary for cell growth and development. SCCAs play critical roles in cellular metabolism1, cell signaling2, and organismal responses to the environment (such as antibiosis3). Because of this, SCCAs can serve as useful indicators of disruptions to cellular metabolism, plant stress responses4,5, and fruit quality6,7. To date, SCCAs have been quantified primarily through chromatographic techniques such as high performance liquid chromatography (HPLC) or gas chromatography-mass spectroscopy (GC-MS). While these methods, are capable of achieving very low limits of detection, they can be expensive, require the derivatization of target SCCAs using caustic and/or toxic reagents, and include lengthy separation runs on the GC or HPLC. Because of this, interest in the use of free zonal capillary electrophoresis (CZE), which does not require sample derivatization, to quantify organic acids has steadily increased8.
Free zonal capillary electrophoresis (CZE) is a chromatographic separation methodology that, due to its high number of theoretical plates, speed, and relative ease-of-use, is increasingly replacing both GC-MS and high-pressure liquid chromatography as an analytical method for the quantification (particularly for quality control purposes) of anions, cations, amino acids, carbohydrates, and short chain carboxylic acids (SCCAs)8,9,10. CZE-based separation of small molecules, including SCCAs, is based two primary principles: the electrophoretic movement of charged ions in an electrical field established across the buffer filling the capillary; and the electro-osmotic movement of the entire buffer system from one end of the capillary to the other, generally towards the negative electrode. In this system, small molecules move towards the negative electrode at varying speeds, with the speed of each molecule determined by the ratio of the net charge of the molecule to the molecular mass. As the movement of each individual molecule in this system is dependent on the charge state of the molecule and the overall rate of electro-osmotic flow (which is itself based on the ion content of the buffer used to fill the capillary), the buffer pH and ionic composition heavily impact the degree to which molecules can be efficiently separated using CZE. Because of this, SCCAs, with their relatively high charge-to-mass ratios, are ideal targets for CZE-based separation. Metabolites separated using CZE can be detected using a variety of methods, including UV absorbance, spectral absorbance (which is generally performed using a photo-diode array [PDA]), and/or mass spectroscopy (CE-MS or CE-MS/MS)8. The diversity of separation and detection methods provided by CZE makes it an extremely flexible and adaptable technique. Because of this, CZE has been increasingly applied as a standard method of analysis in the areas of food safety and quality11,12, pharmaceutical research13, and environmental monitoring13,14. 
Capillary electrophoresis has been used to detect and quantify short chain carboxylic acids for nearly two decades13. The resolving power (particularly for small, charged molecules), short run time, and low per sample cost of CZE analyses make CZE an ideal technique for the separation and quantification of SCCAs13. This method presents a protocol to utilize CZE to measure the concentration of organic acids from plant tissues. Example data was generated through the successful implementation of this protocol to measure the change in organic acid levels in coffee seeds following a secondary fermentation treatment. The protocol details the critical steps and common errors of CZE-based separation of SCCAs, and discusses the means by which this protocol can be successfully applied to quantify SCCAs in additional plant tissues.

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	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Ceramic Moarter and Pestle</td>
			<td style="width:123px;">Coorstek</td>
			<td style="width:119px;">60310</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Beckman Coulter P/ACE MDQ CE system</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">Various</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Glass sample vials</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">033917D</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">1.5 ml microcentrifuge tubes&#160;</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">02-681-5</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:296px;">LC/MS grade water</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">W6-1</td>
			<td style="width:247px;">Milli-Q water (18.2 M&#937;.cm) is also acceptable</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">15 ml glass tube/ Teflon lined cap&#160;</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">14-93331A</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">Parafilm M</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">13-374-12</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">CElixirOA detection Kit pH 5.4&#160;</td>
			<td style="width:123px;">MicroSolv</td>
			<td style="width:119px;">06100-5.4</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">BD Safety-Lok syringes</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">14-829-32</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">17 mm Target Syringe filter, PTFE</td>
			<td style="width:123px;">Fisher Inc.</td>
			<td style="width:119px;">3377154</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">32 Karat, V. 8.0 control software</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">285512</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">capillary electrophoresis (CE) sample vials&#160;</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">144980</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:296px;">caps for CE vials&#160;</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:119px;">144648</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:296px;">Liquid Nitrogen</td>
			<td style="width:123px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:247px;">Liquid nitrogen is available from most facilities services</td>
		</tr>
	</tbody>
</table>

using capillary electrophoresis to quantify organic acids from plant tissue: a test case examining coffea arabica seeds

Carbonsäuren sind organische Säuren einen oder mehrere endständige Carboxyl (COOH) funktionelle Gruppen enthält. Kurzkettige Carbonsäuren (SCCAs; Carbonsäuren mit drei bis sechs Kohlenstoffatomen), wie Malat und Citrat, sind entscheidend für das reibungslose Funktionieren vieler biologischer Systeme, in denen sie in der Zellatmung funktionieren und können als Indikatoren für die Zellgesundheit dienen. In Lebensmitteln kann Gehalt an organischer Säure erhebliche Auswirkungen auf den Geschmack haben, mit einer erhöhten SCCA Ebenen, was zu einem sauren oder &quot;Säure&quot; Geschmack. Wegen dieses Verfahren für die schnelle Analyse von organischen Säurewerte sind von besonderem Interesse für die Lebensmittel- und Getränkeindustrie. Leider jedoch sind die meisten Methoden zur Quantifizierung SCCA sind zeitraubend Protokollen abhängig erfordern analysiert die Derivatisierung der Proben mit gefährlichen Reagenzien, gefolgt durch aufwendige chromatographische und / oder massenspektrometrischen. Dieses Verfahren Informationen ein alternatives Verfahren zum Nachweis und zur Quantifizierung von organic Säuren aus Pflanzenmaterial und Lebensmittelproben frei zonale Kapillarelektrophorese (CZE) mit, manchmal einfach als Kapillarelektrophorese (CE). CZE stellt ein kostengünstiges Verfahren für SCCAs mit einer niedrigen Nachweisgrenze (0,005 mg / ml) zu messen. Dieser Artikel beschreibt die Extraktion und Quantifizierung von SCCAs von Pflanzenproben. Während das Verfahren auf der Messung der SCCAs von Kaffeebohnen vorgesehen konzentriert, kann das Verfahren bereitgestellt, um mehrere auf pflanzlicher Basis Nahrungsmittelmaterialien angewendet werden.

Dieses Protokoll wurde erfolgreich die Auswirkungen der Saatgutbehandlung auf dem SCCA-Gehalt von grünen Kaffeesamen zu messen verwendet. In diesem Experiment wurden die sechs Behandlungen: eine gesättigte Keimsuspension von Leuconostoc - Stamm GCP674 in ihrem Wachstumsmedium pseudomesenteroides (1), eine wässrige Suspension von GCP674 Mikroben in Wasser (2), eine wässrige Lösung von Essigsäure und Milchsäure (0,15 und 0,4 mg / ml) (3), ein M1 - Wachstumsmedium B...

Watch this Scientific Journal Video about Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds at JoVE.com

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Dieser Artikel stellt eine Methode zum Nachweis und zur Quantifizierung von organischen Säuren aus Pflanzenmaterial frei zonale Kapillarelektrophorese. Ein Beispiel für die mögliche Anwendung dieses Verfahrens, um die Auswirkungen einer Nachgärung auf organischen Säure Ebenen in Kaffeesamen zu bestimmen, vorgesehen.

Mit Hilfe der Kapillarelektrophorese organischen Säuren aus Pflanzengewebe zu beziffern: ein Test Untersuchen<em&gt; Coffea arabica</em&gt; Saatgut

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

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Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

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