Determination of five nucleosides by LC‐MS/MS and the application of the method to quantify N⁶‐methyladenosine level in liver messenger ribonucleic acid of an acetaminophen‐induced hepatotoxicity mouse model

Abstract

Ribonucleic acid N6‐methyladenosine methylation plays an important role in a variety of biological processes and diseases. Acetaminophen‐induced hepatotoxicity is one of the major challenges faced by clinicians. To date, the link between N6‐methyladenosine and acetaminophen‐induced hepatotoxicity has not been studied. In this study, a simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of five nucleosides (adenosine, uridine, cytidine, guanosine, and N6‐methyladenosine) in messenger ribonucleic acid. After enzymatic digestion of messenger ribonucleic acid, the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02% formic acid water, and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode. The method was validated over the concentration ranges of 4–800 ng/mL for adenosine, uridine, cytidine, and guanosine and 0.1–20 ng/mL for N6‐methyladenosine. It was successfully applied to the determination of N6‐methyladenosine levels in liver messenger ribonucleic acid in an acetaminophen‐induced hepatotoxicity mouse model and a control group. This study offers a method for the determination of nucleoside contents in epigenetic studies and constitutes the first step toward the investigation of ribonucleic acid methylation in acetaminophen‐induced hepatotoxicity, which will facilitate the elucidation of its mechanism.

UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model

“…Referring to the methods reported, enzymatic digestion of the mRNA was performed [ 14 ]. The mixture sample was made included 100 ng mRNA, 1 μL of nuclease P1 (0.1 U/μL, Takara), and 2 μL of alkaline phosphatase (calf intestine, 1 U/μL, Takara), and the total volume was brought to 30 µL with ultrapure distilled water.…”

“…Among the methylation modifications of mRNA, m 6 A and m 5 C are the most common internal modifications in eukaryotic mRNA, and have been implicated in a variety of biological processes and diseases [ 9 – 11 ]. For example, m 6 A alteration was associated with the progression and metastasis of hepatocellular carcinoma [ 12 , 13 ], and acetaminophen-induced liver injury in mice [ 14 ]. Besides, m 5 C played an important role in regulation of cell stress response, apoptosis, and hepatocellular carcinoma [ 15 , 16 ].…”

“…To the best of our knowledge, some liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods have been reported for the determination of m 6 A or m 5 C in total RNA or mRNA [ 14 , 17 – 22 ]. However, there are some limitations in those methods.…”

“…However, there are some limitations in those methods. For example, the run time of one sample was long [ 20 ], or it need a large amount of mRNA [ 21 , 22 ], or the lower limit of quantification was high [ 14 ]. Therefore, a rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of six nucleosides in mRNA is necessary to be developed for our study.…”

Micro‐flow hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry detects modified nucleosides in the transfer RNA pool of cyanobacteria

“…Other similar studies have been published [10–12]. In addition, monitoring of free modified nucleosides helps to evaluate the quality of pathological animal models and traditional herbs [13, 14]. The term “epitranscriptomics” is now applied to the investigation of the physiological significance of the Spatio‐temporal distribution of post‐transcriptional RNA modification [3, 10, 15, 16].…”