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

作　　者：Wan-Bing Tao Neng-Bin Xie Qing-Yun Cheng Yu-Qi Feng Bi-Feng Yuan 

机构地区：[1]College of Chemistry and Molecular Sciences,Department of Radiation and Medical Oncology,Zhongnan Hospital of Wuhan University,Wuhan University,Wuhan 430071,China [2]School of Public Health,Wuhan University,Wuhan 430071,China [3]Research Center of Public Health,Renmin Hospital of Wuhan University,Wuhan 430060,China [4]Cancer Precision Diagnosis and Treatment and Translational Medicine Hubei Engineering Research Center,Zhongnan Hospital of Wuhan University,Wuhan Research Center for Infectious Diseases and Cancer,Chinese Academy of Medical Sciences,Wuhan 430071,China

出　　处：《Chinese Chemical Letters》2023年第10期98-101,共4页中国化学快报（英文版）

基　　金：supported by the National Key R&D Program of China(Nos.2022YFA0806600,2022YFC3400700);the National Natural Science Foundation of China(Nos.22277093,22074110,21721005).

摘　　要：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.

关 键 词：INOSINE RNA modification CMCT DERIVATIZATION Mass spectrometry Single cell 

分 类 号：O657.63[理学—分析化学] R313[理学—化学]