DNA Methylation Analysis Based On Nanoelectrospray Ionization Mass Spectrometry

DNA methylation is an important epigenetic modification,which is commonly presented as the methylation of the fifth carbon of cytosine in DNA.Mounting researches demonstrated that 5-methylcytosine can regulate gene expression and silencing,and take part in many biological processes,including mammalian development,aging,and carcinogenesis.Therefore,establishing analytical methods to characterize DNA methylation would greatly aid fundamental researches in the field of epigenetics and early diagnosis and prognosis of related diseases.One technique to measure the degree of genomic DNA methylation is mass spectrometry(MS),due to its high selectivity and sensitivity.MS is now widely used in the analysis of DNA methylation,but it still faces many challenges,including large sample consumption,time-consuming procedure,and limited sensitivity to discover new modifications of DNA.In this thesis,three different analytical methods based on Nano ESI-MS were established in order to solve the problems mentioned above.The main development resulting from this thesis are as follows.1.A method sensitive enough to analyze DNA methylation degree of only 100 human cells was established,which was based on on-line sample hydrolysis and Nano ESI-MS.The key strategy employed in this method was to conduct the hydrolysis of genomic DNA directly in the Nano ESI spray emitter,thus minimizing the dilution during sample pretreatment.This method was successfully applied to the DNA methylation analysis of 100 MCF7,MCF10 A,Hep G2,and He La cells within 2 hours.The small sample consumption of this method meant it may be suitable for detection of rare cancer cells in clinical environment or special samples in fundamental researches.2.A method to enable rapid analysis of DNA methylation degree was established,which was based on accelerated derivatization of 2'-deoxycytidine in confined volumes and Nano ESI-MS.BDAPE,a derivative agent for 2'-deoxycytidine and 5-methyl-2'-deoxycytidine,can improve the detection sensitivity of DNA methylation in MS.It was found that the derivatization reaction by BDAPE could be greatly accelerated in confined volumes such as spray droplets.By conducting derivatization in spray droplets,the time needed to finish the reaction can be reduced by 360 folds.Additionally,the concentration of BDAPE needed for confined-volume reactions was much less than that for bulk reactions,thus reducing the matrix effect caused by BDAPE.By combing confinedvolume derivatization reaction with Nano ESI-MS,this method successfully measured the DNA methylation of 6 cell lines,consuming much less time than conventional HPLCMS method.3.A method capable of analyzing the DNA methylation degree at single-cell level was developed,which was based on repeated ion accumulation in linear ion trap and Nano ESI-MS.In this method,commercial mass spectrometer was modified to make the ion trap isolate and accumulate a specific ion repeatedly,thus improving the detection sensitivity of low-concentration molecules.The detection sensitivity of multiple kinds of molecules could be improved by 3-22 folds by this method.The cytosine and 5-methylcytosine hydrolyzed from sample equivalent to 0.2 MCF7 cell could be detected by this method.This method is promising to be used in single-cell analysis of other metabolites and the discovery of new DNA modification.