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 2-week seedlings (fresh weight) and freeze in liquid nitrogen for metabolites extraction. 
	&#8203;CAUTION: Researchers should appropriately wear gloves, protective glasses, and a lab coat to avoid the human-tissue contamination during the materials collection.</li>
</ol>
2 Nucleosides/Nucleotides extraction
<ol>
	<li>Ground 100 mg of frozen plant tissues with 7-8 steel beads in a pre-cold mixer mill for 5 min at a frequency of 60 Hz.</li>
	<li>Prepare the extraction solution, which contains methanol, acetonitrile, and water in a ratio of 2:2:1.</li>
	<li>Resuspend the homogenized materials (including most metabolites but not proteins) with 1 mL of extraction solution.</li>
	<li>Centrifuge the resulting solution at 12,000 x g for 15 min at 4 &#176;C.</li>
	<li>Transfer 0.5 mL of the suspension to a new 1.5 mL tube and freeze in the liquid nitrogen.</li>
	<li>Evaporate the frozen sample in a freeze dyer and resuspend in 0.1 mL of 5% acetonitrile and 95% water.</li>
	<li>Centrifuge the resulting solution (0.1 mL) at 40,000 x g for 10 min at 4 &#176;C. Load the supernatant in a vial for LC-MS/MS measurement.</li>
</ol>
3 LC-MS/MS measurement
<ol>
	<li>Prepare a 10 mM ammonium acetate buffer by dissolving 1.1 g of ammonium acetate in 2 L of double deionized water (Mobile phase A). Adjust the pH to 9.5 by 10% ammonium and acetate acid.</li>
	<li>Prepare 2 L of ultrapure 100% methanol (Mobile phase B1) for nucleosides measurement. Also, prepare 2 L of ultrapure 100% acetonitrile (Mobile phase B2) for nucleotides measurement.</li>
	<li>Inject 0.02 mL of pre-treated metabolites extraction of each sample from step 2.7 into a HPLC system with binary pumps (LC) coupled with a triple quadrupole mass spectrometer (MS). 
	CAUTION: HPLC system employs a C18 column (50 x 4.6 mm, particle size 5 &#181;m; working at 25 &#176;C) buffering with mobile phase A and B1 (Figure 1A) for the nucleosides separation and use a porous graphitic carbon (PGC) column (50 x 4.6 mm, particle size 5 &#181;m; working at 25 &#176;C) with mobile phase A and B2 (Figure 1B) for the nucleotides separation. Each sample was injected three times for the technical replication.</li>
	<li>Program the method as shown in Table 1 for the C18 column, and the method as shown in Table 2 for the PGC column. Set a flow rate of 0.65 mL min-1. 
	NOTE: The mass transitions (Table 3) were monitored by mass spectrometer. The mass spectrum analysis conditions of eight nucleosides and five nucleotides containing canonical ones and modified ones are listed in Table 3.</li>
	<li>Record the peak areas of every target compound (Figure 1).</li>
</ol>
4 Generation of the standard calibration curves
<ol>
	<li>Pool six sample extractions together, which were produced following the description in section 2, and vortex it. Then, aliquot it to six extractions (same volume) again to get each background.</li>
	<li>Add six different concentrations of each standard to these six extractions, respectively, and inject them one by one following step 3.3.</li>
	<li>Record the peak areas of each standard at different concentrations via the mass transitions as described in steps 3.4 and 3.5.</li>
	<li>Plot the peak area against the nominal concentration of each standard to generate a six-point curve. 
	NOTE: The peak areas of nucleosides/nucleotides recorded in the step 3.5 should fall in the range of standard calibration curves.</li>
	<li>Calculate the equation of a straight line for each standard compound: Y = aX + b</li>
</ol>
5 Metabolites&#39; quantification
<ol>
	<li>Calculate the metabolites&#39; contents using the peak area recorded in step 3.5 and the equation from step 4.5.</li>
</ol>

<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>

The authors have no conflict of interest to disclose.

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><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>N&lt;sup&gt;1&lt;/sup&gt;-methyladenosine</td><td>Carbosynth</td><td>NM03697</td><td></td></tr><tr><td>O&lt;sup&gt;6&lt;/sup&gt;-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 ...

plant-sample-preparation-for-nucleosidenucleotide-content-measurement

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Biochemistry

5.1K Views.  Nanjing Agricultural University. A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

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

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

Chemistry

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

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 cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

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

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Environment

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Genetics

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not only provides quantitative data but also permits structural identification of the metabolites present in complex mixtures.
Metabolic profiling by 1H NMR has proven useful in the study of various types of plant scenarios, which include the evaluation of crop&#160;conditions, harvest and post- harvest treatments, metabolic phenotyping, metabolic pathways, gene regulation, identification of biomarkers, chemotaxonomy, quality control, denomination of origin, among others. However, signal overlapping of the large number of resonances with expanded J-coupling multiplicities complicates the spectra analysis and its interpretation, and represents a limitation for classical 1H NMR profiling.
In the last decade, novel NMR broadband homonuclear decoupling techniques through which multiplet signals collapse into single resonance lines - commonly called Pure Shift methods - have been developed to overcome the spectra resolution problem inherent to 1H NMR classical spectra.
Here a step-by-step protocol of the plant extract preparation and the procedure to record optimal Pure Shift PSYCHE and SAPPHIRE-PSYCHE spectra in three different plant matrices - Vanilla plant&#160;leaves, potato tubers (S. tuberosum), and Cape gooseberries (P. peruviana) - is presented. The effect of the gain in resolution in metabolic identification, correlation analysis and multivariate analyses, as compared against classical spectra, is discussed.

This paper presents the use of PSYCHE and SAPPHIRE-PSYCHE in the metabolic profiling of plants and includes detailed procedures for sample preparation and optimal Pure Shift NMR spectra recording. Examples through which the gain in resolution achieved by homonuclear decoupling allows a more comprehensive understanding of the system are discussed.

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not ...

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 and quantify hundreds of thousands of metabolite features. However, the application of these techniques to a large number of samples is subject to more complex interactions, particularly for genome-wide association studies (GWAS). This protocol describes an optimized metabolic workflow, which combines an efficient and fast sample preparation with the analysis of a large number of samples for legume crop species. This slightly modified extraction method was initially developed for the analysis of plant and animal tissues and is based on extraction in methyl tert-butyl ether: methanol solvent to allow the capture of polar and lipid metabolites. In addition, we provide a step-by-step guide for reducing analytical variations, which are essential for the high-throughput evaluation of metabolic variance in GWAS.

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

In this protocol, we present an optimized workflow, which combines an efficient and fast sample preparation of many samples. In addition, we provide a step-by-step guide to reduce analytical variations for high-throughput evaluation of metabolic GWAS studies.

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