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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 deoxyribose^1,2. 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 growth^4,5,6. 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) detector^{4,7,8,9,10,11}. 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/nucleotides^13,14. Several previous studies reported that a HILIC column can be used for nucleosides and nucleotides separation^15,16 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 pseudouridine^1,5,6,18, in plants and Drosophila. Moreover, the method we report here can be used in other organisms as well.

Protokół

1 Plant growth and materials collection

1. 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.
2. 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.
3. 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

1. 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.
2. Prepare the extraction solution, which contains methanol, acetonitrile, and water in a ratio of 2:2:1.
3. Resuspend the homogenized materials (including most metabolites but not proteins) with 1 mL of extraction solution.
4. Centrifuge the resulting solution at 12,000 x g for 15 min at 4 °C.
5. Transfer 0.5 mL of the suspension to a new 1.5 mL tube and freeze in the liquid nitrogen.
6. Evaporate the frozen sample in a freeze dyer and resuspend in 0.1 mL of 5% acetonitrile and 95% water.
7. 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

1. 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.
2. 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.
3. 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.
4. 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.
5. Record the peak areas of every target compound (Figure 1).

4 Generation of the standard calibration curves

1. 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.
2. Add six different concentrations of each standard to these six extractions, respectively, and inject them one by one following step 3.3.
3. Record the peak areas of each standard at different concentrations via the mass transitions as described in steps 3.4 and 3.5.
4. 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.
5. Calculate the equation of a straight line for each standard compound: Y = aX + b

5 Metabolites' quantification

1. Calculate the metabolites' contents using the peak area recorded in step 3.5 and the equation from step 4.5.

Wyniki

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...

Dyskusje

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...

Materiały

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