Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 168, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/61956
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Funding
- Fundamental Research Funds for the Central Universities [LGZD202004]
- National Natural Science Foundation of China [31900907]
- Natural Science Foundation of Jiangsu Province [BK20190528]
- International Centre for Genetic Engineering and Biotechnology [CRP/CHN20-04_EC]
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This study presents a rapid and reliable method for absolute quantification of nucleoside/nucleotide content in plants. By utilizing external standards in HPLC-MS/MS, researchers can easily quantify nucleosides/nucleotides, making it suitable for different plant species.
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.
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