| Autor: |
Lü C; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China.; Beijing Institute of Microchemistry, Beijing 100091, China., Xu H; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., Gao P; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., Huang A; Institute of Beijing Basic Medicine, Academy of Military Medical Sciences, Beijing 100850, China., Qu M; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., He W; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., Wu H; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., Chen J; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., Xu B; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., Guo L; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China., Xie J; Laboratory of Toxicant Analysis, Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China. |
| Abstrakt: |
Accurate detection of endogenous miRNA modifications, such as N6-methyladenosine (m 6 A), 7-methylguanosine (m 7 G), and 5-methylcytidine (m 5 C), poses significant challenges, resulting in considerable uncertainty regarding their presence in mature miRNAs. In this study, we demonstrate for the first time that liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS) nucleoside analysis method is a practical tool for quantitatively analyzing human miRNA modifications. The newly designed liquid-solid two-step hybridization (LSTH) strategy enhances specificity for miRNA purification, while LC-MS/MS offers robust capability in recognizing modifications and sufficient sensitivity with detection limits ranging from attomoles to low femtomoles. Therefore, it provides a more reliable approach compared to existing techniques for revealing modifications in endogenous miRNAs. With this approach, we characterized m 6 A, m 7 G, and m 5 C modifications in miR-21-5p, Let-7a/e-5p, and miR-10a-5p isolated from cultured cells and observed unexpectedly low abundance (<1% at each site) of these modifications. |
