Abundance of Modifications in Mature miRNAs Revealed by LC-MS/MS Method Coupled with a Two-Step Hybridization Purification Strategy.
Anal Chem
; 96(18): 6870-6874, 2024 05 07.
Article
de En
| MEDLINE
| ID: mdl-38648202
ABSTRACT
Accurate detection of endogenous miRNA modifications, such as N6-methyladenosine (m6A), 7-methylguanosine (m7G), and 5-methylcytidine (m5C), 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 m6A, m7G, and m5C 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.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Adénosine
/
Cytidine
/
MicroARN
/
Guanosine
Limites:
Humans
Langue:
En
Journal:
Anal Chem
/
Anal. chem
/
Analytical chemistry
Année:
2024
Type de document:
Article
Pays d'affiliation:
Chine
Pays de publication:
États-Unis d'Amérique