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Mass Spectrometry-based DNA Methylation And Its Oxidation Derivatives Analysis

Posted on:2015-11-07Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y TangFull Text:PDF
GTID:1220330467475146Subject:Analytical Chemistry
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In mammalian DNA,5-methylcytosine (5-mC) is one of the major epigenetic modifications and has been involved in various biological processes. Recent studies in mammals have demonstrated5-mC can be sequentially oxidized to5-hydroxymethylcytosine (5-hmC),5-formylcytosine (5-foC), and5-carboxylcytosine (5-caC) by Ten-eleven translocation (TET) proteins. The oxidation products of5-foC and5-caC can be further recognized and cleaved by thymine-DNA glycosylase (TDG), thereby restoring unmethylated cytosine via the BER pathway. Therefore, active DNA demethylation in mammals could be achieved through an iterative oxidation of5-mC by Ten-eleven translocation (TET) proteins with the generation of three intermediates. Many studies showed a close correlation between active DNA demethylation and organism development/regulation of diverse biological processes.Plant and yeast are belong to eucaryon as well as mammal, and both have the similar gene expression system with mammal, which raised the possible presence of5-mC and its oxidation products (5-hmC,5-foC, and5-caC) in plant or yeast DNA. In previous reports,5-mC and5-hmC were present in plant genomes. Nevertheless, there is no definitive evidence supporting the presence of5-foC and5-caC in genomic DNA of plant or yeast. In addition, method for direct and simultaneous quantification of the four modified cytosines in genomic DNA still lacks due to two problems. First, the content of modified base in DNA is extremely low, and the contents of5-hmC,5-foC, and5-caC decrease successively with1-2order of magnitude in mammalian DNA. Second, owing to the chemical structures of canonical nucleobases are very similar with those of modified nucleobases, bulk regular nucleobases will dramatically interfere the determination of modified nucleobases. Therefore, a highly sensitive and selective method for determining modified cytosines in genomic DNA is requied.Owing to the inherent and high sensitivity and selectivity of gas/liquid chromatography combined with mass spectrometry, we employed gas/liquid chromatography-mass spectrometry combined with chemical derivatization to determine5-mC and its oxidation derivatives in genomic DNA. Chemical derivatization can significantly improve the sensitity and selectivety for the detection of5-mC and its oxidation derivatives in gas/liquid chromatography-mass spectrometry analysis. Then we successfully applied these analytical strategies to determine DNA modifications, which provided solutions for further exploring the physiological function of5-mC and its oxidation derivateves in genomic DNA.1. We developed a highly sensitive method based on gas chromatography/mass spectrometry (GC/MS) and systematically examined the incidence of5-mC in19yeast strains, which represent16yeast species. Our results showed that DNA methylation is widespread in yeast. Furthermore, we found that the5-mC content in yeast varied considerably at different growth stages and DNA methylation inhibitor5-azacytidine could induce a decrease in genome-wide DNA methylation as that in mammalian cells.2. We developed a method to selectively transfer a glucosyl group to the hydroxymethyl moiety of5-hmC and form a more hydrophilic residue (β-glucosyl-5-hydroxymethyl-2’-deoxycytidine,5-gmdC) by using T4(3-glucosyl-transferase. The more hydrophilic5-gmdC can be selectively enriched by using NH2-silica via hydrophilic interaction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which circumvent the problem that the quantification of5-hmC by liquid chromatography-mass spectrometry (LC-MS) frequently suffers from ion suppression due to the presence of unmodified nucleosides. Thus this method can significantly improve the detection sensitivity and accuracy. Using this method, we successfully quantified5-hmC content in genomic DNA of three human cell lines and seven yeast strains. To the best of our knowledge, this is the first report about the existence of5-hmC in the model organism of yeast.3. As for5-foC and5-caC, there is no definitive evidence supporting the presence of these modified nucleobases in plant genomic DNA. Here we developed a chemical derivatization strategy combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method to determine5-formyl-2’-deoxycytidine (5-fodC) and5-carboxyl-2’-deoxycytidine (5-cadC). Derivatization of5-fodC and5-cadC by Girard’s reagents (GirD, GirT, and GirP) significantly increased the detection sensitivities of5-fodC and5-cadC by52-260-fold. Using this method, we first demonstrated the widespread existence of5-fodC and5-cadC in genomic DNA of various plant tissues. Moreover, we found that environmental stresses of drought and salinity can change the contents of5-fodC and5-cadC in plant genomes, suggesting the functional roles of5-fodC and5-cadC in response to environmental stresses.4. We simultaneously derivatized5-methyl-2’-deoxycytidine (5-mdC),5-hydroxymethyl-2’-deoxycytidine (5-hmdC),5-fodC, and5-cadC in genomic DNA with2-bromo-l-(4-dimethylamino-phenyl)-ethanone, which can dramatically improve the retention behavior of these nucleosides in LC and significantly increased the detection sensitivity in ESI-MS. Based on this derivatization, we developed a high sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method to simultaneously determine the four modified cytosines in DNA. Using this method, we successfully quantified5-mC,5-hmC,5-foC, and5-caC in genomic DNA from colorectal carcinoma tumor tissues and relevant tumor adjacent tissues. Moreover, this quantitative report also showed the comparison of the four cytosine modification between genomic DNA from tumor tissues and normal tissues. Compared to normal tissues, the levels of5-hmdC,5-fodC and5-cadC in DNA from tumor tissues decreased, suggesting5-hmdC,5-fodC and5-cadC may play certain functional roles in regulation of cancer formation and development;...
Keywords/Search Tags:DNA methylation, DNA demethylation, cytosine modification, chemicalderivatization, gas chromatography-mass spectrometry, liquid chromatography-tandem massspectrometry
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