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. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+kinase+and+a+glycosylase+catabolize+pseudouridine+in+the+peroxisome+to+prevent+toxic+pseudouridine+monophosphate+accumulation.">A kinase and a glycosylase catabolize pseudouridine in the peroxisome to prevent toxic pseudouridine monophosphate accumulation.</a> The Plant Cell. 32 (3), 722-739 (2020).</li><li>Dahncke, K., 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=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 ...

Research

JoVE Journal

Biochemistry

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization

Large-Scale Multi-Omics Genome-Wide Association Studies Mo-GWAS: Guidelines for Sample Preparation and Normalization

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect ...