Determination Of Cytidine Modifications In Human Body Fluid By Liquid Chromatography-mass Spectrometry Analysis

Both DNA cytosine methylation(5-methyl-2'-deoxycytidine,m~5d C)and RNA cytosine methylation(5-methylcytidine,m~5r C)are important epigenetic marks that play regulatory roles in diverse biological processes.m~5d C and m~5r C can be further oxidized by the ten-eleven translocation(TET)proteins to form 5-hydroxymethyl-2'-deoxycytidine(hm~5d C)and 5-hydroxymethylcytidine(hm~5r C),respectively.2'-O-methyl-5-hydroxymethylcytidine(hm~5r Cm)was recently also identified as a second oxidative metabolite of m~5r C in RNA.Previous studies showed that the dysregulation of cytidine modifications in both DNA and RNA are closely related to a variety of human diseases.These cytidine modifications are generally excreted from cell into urine.If these cytidine modifications exhibit specific features related to certain diseases,determination of the cytidine modifications in urine could be utilized as non-invasive diagnostic of diseases.Liquid chromatography-tandam mass spectrometry(LC-MS/MS)technique owns the strengthes of high selectivity,high sensitivity and strong qualitative and quantitative ability.Hence,LC-MS/MS-based analysis has been widely used in the analysis of trace compounds in clinical and biological samples.However,because of the low abundance of modified nucleosides present in biological fluids and the serious matrix interferences of biological samples,the sensitive detection of modified nucleosides in biological fluids is still challenging.Here,we established a solid-phase extraction in combination with LC-MS/MS analysis for simultaneous detection of these cytidine modifications in human urine samples.We used graphitized carbon black(GCB)cartridges to enrich the nucleosides from urine samples,and the recoveries of all these five cytidine modifications were higher than 92.6%,demonstrating the good performance of GCB cartridges.The relative errors(REs)and intra-and inter-day relative standard deviations(RSDs)were less than 14.0%and 12.0%,respectively,demonstrating that good accuracy and reproducibility were achieved.We analyzed a total of 181 urine samples from cancer patients and healthy controls and for the first time,we reported the presence of hm~5r Cm in human urine.Furthermore,we found that compared to the healthy controls,the contents of hm~5d C,hm~5r C,and hm~5r Cm showed significant increases in urine samples of cancer patients,including lymphoma patients,gastric cancer patients,and esophageal cancer patients.This study indicates that the urinary hydroxylmethylation modifications of hm~5d C,hm~5r C,and hm~5r Cm may serve as potential indicator of cancers.