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

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.