Chemical Labeling – Assisted Mass Spectrometry Analysis for Sensitive Detection of Cytidine Dual Modifications in RNA of Mammals

RNA molecules carry diverse modifications that exert important influences in many cellular processes. In addition to the single modification occurring in either nucleobase or 2' hydroxyl of ribose in RNA, some dual modifications occur in both the nucleobase and 2' hydroxyl of ribose in RNA. 2'-O-methyl-5-methylcytidine (m(5)Cm), the dual modifications of cytidine, was first discovered from the tRNA of archaea. Recent studies identified that 2'-O-methyl-5-hydroxymethylcytidine (hm(5)Cm) and 2'-O-methyl-5-formylcytidine (f(5)Cm) were present in the anticodon of cytoplasmic tRNA of mammals. Similar to the series of single modification of cytidines of 5-methylcytosine (m(5)C), 5-hydroxymethylcytidine (hm(5)C), 5-formylcytidine (f(5)C), and 5-carboxylcytidine (ca(5)C) in nucleic acids, the dual modifications of m(5)Cm, hm(5)Cm, f(5)Cm and 2'-O-methyl-5-carboxylcytidine (ca(5)Cm) may also constitute the series of cytidine modifications in mammals. However, it is normally challenging to detect these modifications because of their low endogenous levels. Here, we established a method by chemical labeling-assisted liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS) analysis for the sensitive and simultaneous determination of all these four cytidine dual modifications, i.e., m(5)Cm, hm(5)Cm, f(5)Cm and ca(5)Cm. Three different labeling reagents (2-bromo-1-(3,4-dimeth oxyphenyl)- ethanone, BDMOPE; 2-bromo 1 (4 methoxyphenyl)-ethanone, BMOPE; 2-bromo-1-(4-diethylaminophenyl)-ethanone, BDEPE) were used for the chemical labeling. The results showed that the detection sensitivities of m(5)Cm, hm(5)Cm, f(5)Cm and ca(5)Cm increased up to 462 folds after chemical labeling. With the developed method, we achieved the simultaneous detection of m(5)Cm, hm(5)Cm and f(5)Cm in RNA of mammals. In addition, we found these cytidine dual modifications mainly exist in small RNA (<200 nt) and barely detected in other types of RNA. Moreover, we found that the levels of m(5)Cm in RNA of human lung carcinoma tissues significantly increased, while hm(5)Cm and f(5)Cm significantly decreased compared to tumor adjacent normal tissues. The significant changes of m(5)Cm, hm(5)Cm and f(5)Cm levels may serve as indicator for the detection and prognosis of lung cancer. (C) 2019 Elsevier B.V. All rights reserved.