Summary
A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.
Abstract
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.
Introduction
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 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.
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Protocol
1 Plant growth and materials collection
- 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.
- Incubate the plates containing Arabidopsis seeds under dark at 4 °C for 48 h, and then transfer them into a controlled growth chamber under 16 h light of 55 µmol m-2 s-1 at 22 °C and 8 h dark at 20 °C.
- Harvest 100 mg of 2-week seedlings (fresh weight) and freeze in liquid nitrogen for metabolites extraction.
CAUTION: Researchers should appropriately wear gloves, protective glasses, and a lab coat to avoid the human-tissue contamination during the materials collection.
2 Nucleosides/Nucleotides extraction
- 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.
- Prepare the extraction solution, which contains methanol, acetonitrile, and water in a ratio of 2:2:1.
- Resuspend the homogenized materials (including most metabolites but not proteins) with 1 mL of extraction solution.
- Centrifuge the resulting solution at 12,000 x g for 15 min at 4 °C.
- Transfer 0.5 mL of the suspension to a new 1.5 mL tube and freeze in the liquid nitrogen.
- Evaporate the frozen sample in a freeze dyer and resuspend in 0.1 mL of 5% acetonitrile and 95% water.
- Centrifuge the resulting solution (0.1 mL) at 40,000 x g for 10 min at 4 °C. Load the supernatant in a vial for LC-MS/MS measurement.
3 LC-MS/MS measurement
- 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.
- 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.
- 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 µm; working at 25 °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 µm; working at 25 °C) with mobile phase A and B2 (Figure 1B) for the nucleotides separation. Each sample was injected three times for the technical replication. - 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. - Record the peak areas of every target compound (Figure 1).
4 Generation of the standard calibration curves
- 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.
- Add six different concentrations of each standard to these six extractions, respectively, and inject them one by one following step 3.3.
- Record the peak areas of each standard at different concentrations via the mass transitions as described in steps 3.4 and 3.5.
- 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. - Calculate the equation of a straight line for each standard compound: Y = aX + b
5 Metabolites' quantification
- Calculate the metabolites' contents using the peak area recorded in step 3.5 and the equation from step 4.5.
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Representative Results
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 abundance of the product ion of 150 m/z, the mass transition of 282.1 to 150 (m/z) is selected for the N1-methyladenosine quantification. In addition, the retention time (RT) of target peak (Figure 3B) is 7.05 min, which is same as the RT of N1-methyladenosine standard (Figure 3A). Considering the data mentioned above, we demonstrate that wild type seedlings contain in vivo N1-methyladenosine pool.
A concentration series of N1-methyladenosine standards (0, 1, 2.5, 5, 10, and 50 ng / mL) was added into six sample extractions produced following steps 4.1 and 4.2, respectively (Figure 4A). 0.02 mL of each standard samples was injected into the LC-MS/MS, and the increased peak areas of N1-methyladenosine were plotted against the nominal concentrations of N1-methyadenosine standards. The equation of the straight line is Y = 0.0004X - 0.163 (Figure 4B).
Three replicates of Col-0 seedlings were extracted and pre-treated as described above. The peak area of N1-methyladenosine in these three samples were recorded as 8,659, 12,147, and 12,711. Considering the five times enrichment during the extraction (see steps 2.5 and 2.6) and using the equation Y = 0.0004X - 0.163, N1-methyladenosine concentration were calculated in three wild type lines to be 0.66, 0.94, and 0.98 ng / mL, respectively. Hence, 100 mg of each wild type seedlings were used for extraction and resuspended in 1 mL extraction buffer. Therefore, 8.6 ± 1.7 ng of N1-methyladenosine was quantified in 1 g of 2-week-old Arabidopsis wild type seedlings.
| Time | Flow rate (mL min-1) | Mobile phase A (%) | Mobile phase B (methanol %) |
| 0 | 0.65 | 95 | 5 |
| 2 | 0.65 | 95 | 5 |
| 5.5 | 0.65 | 85 | 15 |
| 9.5 | 0.65 | 15 | 85 |
| 11 | 0.65 | 15 | 85 |
| 11.1 | 0.65 | 95 | 5 |
| 20 | 0.65 | 95 | 5 |
Table 1: The method for the C18 column. Schematic representation of solvent changes for the equilibration of C18 column. Mobile phase A = 10 mM ammonium acetate, pH 9.5. Mobile phase B = 100% methanol.
| Time | Flow rate (mL min-1) | Mobile phase A (%) | Mobile phase B (acetonitrile %) |
| 0 | 0.65 | 90 | 10 |
| 9 | 0.65 | 0 | 100 |
| 10.4 | 0.65 | 0 | 100 |
| 10.6 | 0.65 | 90 | 10 |
| 21 | 0.65 | 90 | 10 |
Table 2: The method for the PGC column. Schematic representation of solvent changes for the equilibration of PGC column. Mobile phase A = 10mM ammonium acetate, pH 9.5. Mobile phase B = 100% acetonitrile.
| Nucleosides/nucleotides | Mass transition (m/z) | Polarity | Fragmentor | Collision energy (eV) | Cell Accelerator voltage | Retention time (min) | Retention Window (min) | Monitoring Mode | |
| Precursor ion | Product ion | ||||||||
| adenosine | 268.1 | 136 | Positive | 86 | 15 | 4 | 9.3 | 1.5 | MRM |
| N1-methyladenosine | 282.12 | 150 | Positive | 88 | 19 | 4 | 8.1 | 1.5 | MRM |
| guanosine | 284.1 | 135 | Positive | 90 | 45 | 4 | 7.9 | 1.5 | MRM |
| O6-methylguanosine | 298.12 | 166 | Positive | 68 | 19 | 4 | 9.8 | 1.5 | MRM |
| inosine | 269.1 | 136.9 | Positive | 55 | 14 | 4 | 7.2 | 1.5 | MRM |
| uridine | 245.21 | 133 | Positive | 85 | 14 | 4 | 3.7 | 1.5 | MRM |
| pseudouridine | 245.21 | 125 | Positive | 68 | 15 | 4 | 1.9 | 1.5 | MRM |
| cytidine | 244.2 | 112 | Positive | 150 | 10 | 4 | 2.6 | 1.5 | MRM |
| AMP | 348.07 | 136 | Positive | 111 | 17 | 4 | 11.8 | 2 | MRM |
| GMP | 364.07 | 152 | Positive | 80 | 45 | 4 | 11.6 | 2 | MRM |
| IMP | 348.9 | 137 | Positive | 79 | 15 | 4 | 10.9 | 2 | MRM |
| UMP | 325.01 | 212.9 | Positive | 98 | 3 | 4 | 9.4 | 2 | MRM |
| CMP | 324 | 112 | Positive | 90 | 12 | 4 | 9.1 | 2 | MRM |
Table 3: MS analysis conditions of nucleosides and nucleotides detected by mass spectrometer. The precursor ion and product ion of eight nucleosides and six nucleotides are listed here and can be monitored by MS for compound identification and quantification.
Figure 1: The chromatographic peaks of eight nucleosides and five nucleotides. The separation profiles of eight nucleosides by the C18 column (A), and five nucleotides by the PGC column (B). Please click here to view a larger version of this figure.
Figure 2: Identification of N1-methyladenosine by mass transition. MS/MS spectra of precursor ion m/z 282.1 and product ions m/z 150 and m/z 133 detected from N1-methyladenosine standard (A) and Col-0 samples (B). Please click here to view a larger version of this figure.
Figure 3: The chromatographic peak of N1-methyladenosine. Mass transition of 282.1 to 150 was monitored for N1-methyladenosine quantification. The retention times of N1-methyladenosine peaks in the standard and sample measurement were similar. Please click here to view a larger version of this figure.
Figure 4: Generation of the N1-methyladenosine standard curve. (A) Six different concentrations of N1-methyladenosine were added into six sample extraction matrixes, respectively. And the resulted increase peak areas were recorded. (B) The calibration curve of N1-methyladenosine. Please click here to view a larger version of this figure.
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Discussion
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 spectrum for nucleosides/nucleotides detection. Taking N1-methyladenosine as an example, this method could detect as low as 0.02 ng standard, and the accuracy of the calibration curve is quite high (R2 = 0.999; Figure 4B). Compared with the HPLC method, an MS-based protocol provides much better detection limit and accuracy. More importantly, this method can be easily performed by researchers in a biological laboratory that has a LC-MS/MS. Moreover, it can also be used for the identification of other structures known metabolites in plants.
For the in vivo absolute quantification of nucleosides/nucleotides content, commercial standard chemicals are required. They produce the straight standard curves, which allow to calculate the target metabolites in samples through peak areas recorded by mass spectrometry. It is important that the range of peak areas in standard calibration curves should cover the peak area of target metabolite read in MS. Moreover, a concentration series of standards should be added into the sample extractions but not dissolved in water for calibration curve generation. This is because it will avoid the matrix effect, which is tremendously significant for quantification accuracy.
The method described here provides a powerful tool for nucleosides/nucleotides quantification. Its application can extend to all plants and even other organisms. The whole procedure of samples' pre-treatment needs to stay cold and fast to avoid metabolites degradation, although the extraction buffer contains 80% organic chemicals, which could precipitate most of the proteins (enzymes). However, this method is not suitable for unknown target identification. The identification and quantification of target chemical in this method largely depends on the commercial chemical standards. Another limitation of this method is that the measurement of nucleosides and nucleotides has to be done separately by employing a C18 column and a PGC column, respectively. It is because that the performance of the C18 column, although, is more stable and reproducible than PGC column, the latter could especially distinguish nucleotides much better (Figure 1B).
In conclusion, the presented method allows in vivo quantification of nucleosides/nucleotides in plants. From seedlings growth to obtaining the final results, the experiments can be completed within 3 weeks. Complete samples pre-treatments and LC-MS/MS analyses take about 2 days for a set of 10 to 20 samples.
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Disclosures
The authors have no conflict of interest to disclose.
Acknowledgments
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.
Materials
| Name | Company | Catalog Number | Comments |
| acetonitrile | Sigma-Aldrich | 1000291000 | |
| adenosine | Sigma-Aldrich | A9251-1G | |
| ammonium acetate | Sigma-Aldrich | 73594-100G-F | |
| AMP | Sigma-Aldrich | 01930-5G | |
| CMP | Sigma-Aldrich | C1006-500MG | |
| cytidine | Sigma-Aldrich | C122106-1G | |
| GMP | Sigma-Aldrich | G8377-500MG | |
| guanosine | Sigma-Aldrich | G6752-1G | |
| Hypercarb column | Thermo Fisher Scientific GmbH | 35005-054630 | |
| IMP | Sigma-Aldrich | 57510-5G | |
| inosine | Sigma-Aldrich | I4125-1G | |
| methanol | Sigma-Aldrich | 34860-1L-R | |
| N1-methyladenosine | Carbosynth | NM03697 | |
| O6-methylguanosine | Carbosynth | NM02922 | |
| Murashige and Skoog Medium | Duchefa Biochemie | M0255.005 | |
| Polaris 5 C18A column | Agilent Technologies | A2000050X046 | |
| pseudouridine | Carbosynth | NP11297 | |
| UMP | Sigma-Aldrich | U6375-1G | |
| uridine | Sigma-Aldrich | U3750-1G |
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