论文部分内容阅读
Background: 5-methylcytosine (5-mC) represents an important epigenetic modification involved in development and is frequently altered in cancer (1,2).5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) in an enzymatic process.5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons,but the distribution of 5-hmC in human liver tumor and matched control tissues has not been rigorously explored (3).Methods: We developed an online trapping-capillary hydrophilic interaction liquid chromatography (cHILIC)-in source fragmentation-tandem mass spectrometry (MS/MS) system for quantifying 5-mC and 5-hmC in genomic DNA from hepatocellular carcinoma (HCC) tumor tissues and relevant tumor adjacent tissues.A polymer-based hydrophilic monolithic column was prepared and used for the separation of twelve nucleosides by cHILIC coupled with online trapping system.Limits of detection and quantification (LODs and LOQs),recovery and imprecision of the method were determined.Results: LODs for 5-mC and 5-hmC were 0.06 and 0.19 fmol,respectively,which were,to the best of our knowledge,the lowest compared to other previously reported methods using mass spectrometry.The imprecision and recovery of the cHILIC-ESI-qTOF-MS/MS method were determined with the relative standard deviations (RSDs) and relative errors (REs) being less than 14.9% and 15.8%,respectively.There was a 4-5 fold lower 5-hmC content in HCC tumor tissues compared to tumor adjacent tissues.In addition,5-hmC content highly correlated with tumor stage (Tumor nodes metastasis,p = 0.0002; Barcelona clinic liver cancer,p = 0.0003).Conclusions: The marked depletion of 5-hmC may have profound effects on epigenetic regulation in HCC and could be a potential biomarker for the early detection and prognosis of HCC.