Sensitive determination of inosine RNA modification in single cell by chemical derivatization coupled with mass spectrometry analysis

Abstract

Inosine is a vital RNA modification across three kingdoms of life. It has been demonstrated that inosine plays important roles in modulation of the fate of RNAs. In the current study, we developed a highly sensitive method to determine inosine in a single cell by N-cyclohexyl-N’-β-(4-methylmorpholinium)ethylcarbodiimide p-toluenesulfonate (CMCT) derivatization in combination with mass spectrometry analysis. The results showed that the detection sensitivity of inosine was increased by 556-fold after CMCT derivatization, with the limit of detection (LOD) being 4.5 amol. With the established method, we could detect inosine from 13.0 pg of total RNA of HEK293T cells. Meanwhile, inosine in RNA from a single cell could also be clearly detected due to the improved detection sensitivity. Moreover, we found the level of inosine in RNA of sleep-deprived mice was significantly increased compared to the control mice, indicating that inosine is associated with sleep behavior and might be a potential indicator of sleep disorder. Taken together, the chemical derivatization coupled with mass spectrometry analysis offers a valuable tool in determination of endogenous RNA modifications in a single cell, which should benefit the functional study of RNA modification in rare clinical samples.