Chemical labeling – Assisted mass spectrometry analysis for sensitive detection of cytidine dual modifications in RNA of mammals

Abstract 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 (m5Cm), the dual modifications of cytidine, was first discovered from the tRNA of archaea. Recent studies identified that 2′-O-methyl-5-hydroxymethylcytidine (hm5Cm) and 2′-O-methyl-5-formylcytidine (f5Cm) were present in the anticodon of cytoplasmic tRNA of mammals. Similar to the series of single modification of cytidines of 5-methylcytosine (m5C), 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C), and 5-carboxylcytidine (ca5C) in nucleic acids, the dual modifications of m5Cm, hm5Cm, f5Cm and 2′-O-methyl-5-carboxylcytidine (ca5Cm) 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., m5Cm, hm5Cm, f5Cm and ca5Cm. 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 m5Cm, hm5Cm, f5Cm and ca5Cm increased up to 462 folds after chemical labeling. With the developed method, we achieved the simultaneous detection of m5Cm, hm5Cm and f5Cm in RNA of mammals. In addition, we found these cytidine dual modifications mainly exist in small RNA (