Name: plant sample preparation for nucleoside/nucleotide content measurement with an hplc-ms/ms
Uploaded: 2021-02-24T14:00:00
Description: Watch this Scientific Journal Video about Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS at JoVE.com

This work was financially supported by the Fundamental Research Funds for the Central Universities (KJQN202060), the National Natural Science Foundation of China (31900907), the Natural Science Foundation of Jiangsu Province (BK20190528), the International Centre for Genetic Engineering and Biotechnology (CRP/CHN20-04_EC) to M.C., and the Fundamental Research Funds for the Central Universities (LGZD202004) to X.L.

1 Plant growth and materials collection
<ol>
	<li>Ensure that Arabidopsis seeds are sterilized in 70% ethanol for 10 min and sowed on the agar plates, which were prepared with one-half-strength Murashige and Skoog nutrients.</li>
	<li>Incubate the plates containing Arabidopsis seeds under dark at 4 &#176;C for 48 h, and then transfer them into a controlled growth chamber under 16 h light of 55 &#181;mol m-2 s-1 at 22 &#176;C and 8 h dark at 20 &#176;C.</li>
	<li>Harvest 100 mg of...

<ol>
	<li>Liu, B., Winkler, F., Herde, M., Witte, C. -. P., Gro&#223;hans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+link+between+deoxyribonucleotide+metabolites+and+embryonic+cell-cycle+control.">A link between deoxyribonucleotide metabolites and embryonic cell-cycle control.</a> Current Biology. 29 (7), 1187-1192 (2019).</li><li>Zrenner, R., Stitt, M., Sonnewald, U., Boldt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrimidine+and+purine+biosynthesis+and+degradation+in+plants.">Pyrimidine and purine biosynthesis and degradation in plants.</a> Annual Review of Plant Biology. 57, 805-836 (2006).</li><li>Witte, C. -. P., Herde, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+metabolism+in+plants.">Nucleotide metabolism in plants.</a> Plant Physiology. 182 (1), 63-78 (2020).</li><li>Chen, M., Herde, M., Witte, C. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+the+nine+cytidine+deaminase-like+genes+in+Arabidopsis,+eight+are+pseudogenes+and+only+one+is+required+to+maintain+pyrimidine+homeostasis+in+vivo.">Of the nine cytidine deaminase-like genes in Arabidopsis, eight are pseudogenes and only one is required to maintain pyrimidine homeostasis in vivo.</a> Plant Physiology. 171 (2), 799-809 (2016).</li><li>Chen, M., 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=m6A+RNA+degradation+products+are+catabolized+by+an+evolutionarily+conserved+N6-methyl-AMP+deaminase+in+plant+and+mammalian+cells.">m6A RNA degradation products are catabolized by an evolutionarily conserved N6-methyl-AMP deaminase in plant and mammalian cells.</a> The Plant Cell. 30 (7), 1511-1522 (2018).</li><li>Chen, M., Witte, C. -. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+purine+nucleoside+catabolism+employs+a+guanosine+deaminase+required+for+the+generation+of+xanthosine+in+Arabidopsis.">Plant purine nucleoside catabolism employs a guanosine deaminase required for the generation of xanthosine in Arabidopsis.</a> The Plant Cell. 25 (10), (2013).</li><li>Jung, B., 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=Uridine-ribohydrolase+is+a+key+regulator+in+the+uridine+degradation+pathway+of+Arabidopsis.">Uridine-ribohydrolase is a key regulator in the uridine degradation pathway of Arabidopsis.</a> The Plant Cell. 21 (3), 876-891 (2009).</li><li>Jung, B., Hoffmann, C., Moehlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+nucleoside+hydrolases+involved+in+intracellular+and+extracellular+degradation+of+purines.">Arabidopsis nucleoside hydrolases involved in intracellular and extracellular degradation of purines.</a> Plant Journal. 65 (5), 703-711 (2011).</li><li>Riegler, H., Geserick, C., Zrenner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+thaliana+nucleosidase+mutants+provide+new+insights+into+nucleoside+degradation.">Arabidopsis thaliana nucleosidase mutants provide new insights into nucleoside degradation.</a> New Phytologist. 191 (2), 349-359 (2011).</li><li>Zrenner, R., 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=A+functional+analysis+of+the+pyrimidine+catabolic+pathway+in+Arabidopsis.">A functional analysis of the pyrimidine catabolic pathway in Arabidopsis.</a> New Phytologist. 183 (1), 117-132 (2009).</li><li>Hauck, O. K., 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=Uric+acid+accumulation+in+an+Arabidopsis+urate+oxidase+mutant+impairs+seedling+establishment+by+blocking+peroxisome+maintenance.">Uric acid accumulation in an Arabidopsis urate oxidase mutant impairs seedling establishment by blocking peroxisome maintenance.</a> The Plant Cell. 26 (7), 3090-3100 (2014).</li><li>Qu, 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=Comparative+analysis+of+nucleosides,+nucleobases,+and+amino+acids+in+different+parts+of+Angelicae+Sinensis+Radix+by+ultra+high+performance+liquid+chromatography+coupled+to+triple+quadrupole+tandem+mass+spectrometry.">Comparative analysis of nucleosides, nucleobases, and amino acids in different parts of Angelicae Sinensis Radix by ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry.</a> Journal of Separation Science. 42 (6), 1122-1132 (2019).</li><li>Zong, S. -. Y., 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=Fast+simultaneous+determination+of+13+nucleosides+and+nucleobases+in+Cordyceps+sinensis+by+UHPLC-ESI-MS/MS.">Fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis by UHPLC-ESI-MS/MS.</a> Molecules. 20 (12), 21816-21825 (2015).</li><li>Moravcov&#225;, D., 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=Separation+of+nucleobases,+nucleosides,+and+nucleotides+using+two+zwitterionic+silica-based+monolithic+capillary+columns+coupled+with+tandem+mass+spectrometry.">Separation of nucleobases, nucleosides, and nucleotides using two zwitterionic silica-based monolithic capillary columns coupled with tandem mass spectrometry.</a> Journal of Chromatography. A. 1373, 90-96 (2014).</li><li>Guo, S., 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=Hydrophilic+interaction+ultra-high+performance+liquid+chromatography+coupled+with+triple+quadrupole+mass+spectrometry+for+determination+of+nucleotides,+nucleosides+and+nucleobases+in+Ziziphus+plants.">Hydrophilic interaction ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry for determination of nucleotides, nucleosides and nucleobases in Ziziphus plants.</a> Journal of Chromatography. A. 1301, 147-155 (2013).</li><li>Seifar, R. M., 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=Simultaneous+quantification+of+free+nucleotides+in+complex+biological+samples+using+ion+pair+reversed+phase+liquid+chromatography+isotope+dilution+tandem+mass+spectrometry.">Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry.</a> Analytical Biochemistry. 388 (2), 213-219 (2009).</li><li>Baccolini, C., Witte, C. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMP+and+GMP+catabolism+in+Arabidopsis+converge+on+xanthosine,+which+is+degraded+by+a+nucleoside+hydrolase+heterocomplex.">AMP and GMP catabolism in Arabidopsis converge on xanthosine, which is degraded by a nucleoside hydrolase heterocomplex.</a> The Plant Cell. 31 (3), 734-751 (2019).</li></ol>

Organisms contain various nucleosides/nucleotides, including canonical and aberrant ones. However, the origin and metabolic endpoints of them, especially modified nucleosides, are still obscure. Furthermore, the current understanding of the function and homeostasis of nucleosides/nucleotides metabolism remain to be explored and expanded. To investigate them, a precise and gold-standard method for these metabolites identification and quantification needs to be employed. Here, we described a protocol using the mass spectru...

Nucleosides/Nucleotides are central metabolic components in all living organisms, which are the precursors for nucleic acids and many coenzymes, such as nicotinamide adenine dinucleotide (NAD), and important in the synthesis of macromolecules such as phospholipids, glycolipids, and polysaccharides. Structurally, nucleoside contains a nucleobase, which can be an adenine, guanine, uracil, cytosine, or thymine, and a sugar moiety, which can be a ribose or a deoxyribose1,2. Nucleotides have up to three phosphate groups binding to the 5-carbon position of the sugar moiety of the nucleosides3. The metabolism of nucleotides in plants is essential for seed germination and leaf growth4,5,6. To better understand their physiological roles in plant development, the methods for the absolute quantification of different nucleosides/nucleotides in vivo should be established.
One of the most commonly used approaches to measure nucleosides/nucleotides employs a high-performance liquid chromatography (HPLC) coupled with an ultraviolet-visible (UV-VIS) detector4,7,8,9,10,11. In 2013, using HPLC, Dahncke and Witte quantified several types of the nucleosides in Arabidopsis thaliana7. They identified an enhanced guanosine content in a T-DNA insertion mutant targeting in the guanosine deaminase gene compared to the wild-type plant. Another pyrimidine nucleoside, cytidine, was also quantitatively detected in plants employing this method, which resulted in the identification of a bona fide cytidine deaminase gene4. Based on the UV detector, this method, however, cannot easily distinguish the nucleosides which have similar spectrums and retention times, e.g., guanosine or xanthosine. The detection limit of HPLC method is relatively high, therefore, it is frequently used for the measurement of high content of nucleosides in vivo, such as cytidine, uridine, and guanosine.
In addition, gas chromatography coupled to mass spectrometry (GC-MS) can also be used in nucleoside measurement. Benefiting from it, Hauck et. al. successfully detected uridine and uric acid, which is a downstream metabolite of nucleoside catabolic pathway, in the seeds of A. thaliana12. However, GC is normally used to separate volatile compounds but not suitable for the thermally labile substances. Therefore, a liquid chromatography coupled to mass spectrometry (LC-MS/MS) is probably a more suitable and accurate analytical technique for the&#160;in vivo identification, separation, and quantification of the nucleosides/nucleotides13,14. Several previous studies reported that a HILIC column can be used for nucleosides and nucleotides separation15,16 and isotopically labeled internal standards were employed for the compound quantification17. However, both components are relatively expensive, especially the commercial isotope-labeled standards. Here, we report an economically applicable LC-MS/MS approach for nucleosides/nucleotides measurement. This method has been already successfully used for the quantitation of diverse nucleosides/nucleotides, including ATP, N6-methyl-AMP, AMP, GMP, uridine, cytidine, and pseudouridine1,5,6,18, in plants and Drosophila. Moreover, the method we report here can be used in other organisms as well.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>1000291000</td>
			<td></td>
		</tr>
		<tr>
			<td>adenosine</td>
			<td>Sigma-Aldrich</td>
			<td>A9251-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>73594-100G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP</td>
			<td>Sigma-Aldrich</td>
			<td>01930-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>CMP</td>
			<td>Sigma-Aldrich</td>
			<td>C1006-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>cytidine</td>
			<td>Sigma-Aldrich</td>
			<td>C122106-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP</td>
			<td>Sigma-Aldrich</td>
			<td>G8377-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>guanosine</td>
			<td>Sigma-Aldrich</td>
			<td>G6752-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypercarb column</td>
			<td>Thermo Fisher Scientific GmbH</td>
			<td>35005-054630</td>
			<td></td>
		</tr>
		<tr>
			<td>IMP</td>
			<td>Sigma-Aldrich</td>
			<td>57510-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>inosine</td>
			<td>Sigma-Aldrich</td>
			<td>I4125-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860-1L-R</td>
			<td></td>
		</tr>
		<tr>
			<td>N1-methyladenosine</td>
			<td>Carbosynth</td>
			<td>NM03697</td>
			<td></td>
		</tr>
		<tr>
			<td>O6-methylguanosine</td>
			<td>Carbosynth</td>
			<td>NM02922</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog Medium</td>
			<td>Duchefa Biochemie</td>
			<td>M0255.005</td>
			<td></td>
		</tr>
		<tr>
			<td>Polaris 5 C18A column</td>
			<td>Agilent Technologies</td>
			<td>A2000050X046</td>
			<td></td>
		</tr>
		<tr>
			<td>pseudouridine</td>
			<td>Carbosynth</td>
			<td>NP11297</td>
			<td></td>
		</tr>
		<tr>
			<td>UMP</td>
			<td>Sigma-Aldrich</td>
			<td>U6375-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3750-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

plant sample preparation for nucleoside/nucleotide content measurement with an hplc-ms/ms

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

Here, we show the identification and quantification of N1-methyladenosine, a known modified nucleoside, in 2-week-old Arabidopsis wild type (Col-0) seedlings as an example. Mass spectrometry profile indicates that the product ions generated from the N1-methyladenosine standard are 150 m/z and 133 m/z (Figure 2A), and the same profile is also observed in Col-0 extraction (Figure 2B). Due to high abun...

Watch this Scientific Journal Video about Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS at JoVE.com

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

Nucleosides and nucleotides are central metabolic components in all living organisms. The metabolism of them is required for cell development. This approach provides a powerful tool for these metabolites'quantification.This method allows rapid and accurate quantification of nucleosides and nucleotides in plants. Complete sample pre-treatments and LC-MS/MS analyses take about two days for a set of 10 to 20 samples. This method can be applied to all plants and even other organisms.Demonstrating the procedure will be Changhua Zhu, an associate professor from the Chen Laboratory. To grow the plants, ensure that Arabidopsis seeds are sterilized in 70%ethanol for 10 minutes and sowed on agar plates with 1/2 strength Murashige and Skoog nutrients. Incubate the plates in the dark at four degrees Celsius for 48 hours, then transfer them to a controlled growth chamber under 16 hours of light at 22 degrees Celsius and eight hours of dark at 20 degrees Celsius.Harvest 100 milligrams of two-week seedlings and freeze them in liquid nitrogen for metabolite extraction. Grind 100 milligrams of frozen plant tissues with seven to eight steel beads in a pre-cooled mixer mill for five minutes at a frequency of 60 Hertz. Prepare the extraction solution, which contains methanol, acetonitrile, and water at a ratio of two-to-two-to-one.Resuspend the homogenized materials with one milliliter of the extraction solution. Centrifuge the resulting solution at 12, 000 XG for 15 minutes at four degrees Celsius, then transfer 0.5 milliliters of the suspension to a new 1.5 milliliter tube and freeze it in liquid nitrogen. Evaporate the frozen sample in a freeze dryer and resuspend it in 0.5 milliliter of 5%acetonitrile and 95%water.Centrifuge the resulting solution at 4, 000 XG for 10 minutes at four degrees Celsius. Load the supernatant into a vial for LC-MS/MS measurement. Prepare a 10 millimolar ammonium acetate buffer by dissolving 1.5 grams of ammonium acetate in two liters of double deionized water.Adjust the pH to 9.5 with 10%ammonium and acetate acid. Prepare two liters of ultrapure 100%methanol for nucleosides measurement, then prepare two liters of ultrapure acetonitrile for nucleotides measurement. Inject 0.02 milliliters of the pretreated metabolite extraction of each previously prepared sample into an HPLC system with binary pumps coupled with a triple quadruple mass spectrometer.To generate the standard calibration curves, pool six sample extractions together and vortex the mixture, then aliquot it to six extractions again to obtain each background. Add six different concentrations of each standard to these six extractions respectively and inject them one by one into the HPLC system. Record the peak areas of each standard at different concentrations via the mass transitions.This protocol was used to identify and quantify N1-methyladenosine, a known modified nucleoside, in two-week-old Arabidopsis wild-type seedlings. The mass spectrometry profile indicated that the product ions generated from the N1-methyladenosine standard are at 150 and 133 MZ ratios. And the same profile was observed in the Colombia zero extraction.Due to the high abundance of the product ion of 150 MZ, the mass transition of 282.1 to 150 was selected for the N1-methyladenosine identification. The retention time of the target peak was 7.05 minutes. Same as the retention time of N1-methyladenosine standard.A concentration series of N1-methyladenosine standards was added into six sample extractions. The standard samples were injected into the LC-MS/MS and the increased peak areas of N1-methyladenosine were plotted against the nominal concentrations of the standards. The six equal background extractions are important for making an accurate and repeatable calibration curve for the quantification.Our method can be applied to explore metabolic pathways in plants. By comparing the metabolite profiles between wild-type and loss-of-function mutants, the metabolic flux could be drawn.

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