Mass spectrometric study of genetic and epigenetic DNA modifications.

摘要

In this dissertation, I focus on the development of novel MS-based strategies to identify and quantify DNA lesions formed in isolated DNA and in cells to monitor the progression of enzymatic reactions in vitro and glyoxal or methylglyoxal exposure in vivo. In addition, a combined SILAC, one-step affinity purification and LC-MS/MS approach was employed for identifying systematically cellular proteins capable of binding to 6- thioguanine-containing duplex DNA.;In Chapter 2, a stable isotope dilution coupled with LC-MS/MS/MS method was developed to quantify accurately DNA advance glycation end products (AGEs) including N2-carboxymethyl-2'-deoxyguanosine (N2-CMdG), and two diastereomers of N2-(1-carboxyethyl)-2'-deoxyguanosine ( N2-CEdG) induced by hyperglycemia in calf thymus DNA, cellular DNA, rat and mouse tissues and human blood samples. The results showed thatN22-CMdG and N2-CEdG were stable and the level of N2-CMdG and two diastereomers of N2-CEdG were higher in the liver tissues of diabetic mice than those of the healthy control. This work shows that N2-CMdG and N2-CEdG might serve as molecular biomarkers for monitoring glyoxal and methylglyoxal exposure.;In Chapter 3, I established a novel restriction enzyme digestion coupled with LC-MS/MS method to investigate the effect of 6-thioguanine on the HpaII- and DNMT1-mediated methylation of cytosine in synthetic duplex DNA. Moreover, the level of global cytosine methylation in different leukemia cell lines upon 6-thioguanine treatment was evaluated by an offline HPLC method. These results provided important new knowledge about the antileukemic effects of 6-thioguanine.;In Chapter 4, 6-thioguanine and its metabolite, S6-methylthio-2'-deoxyguanosine in genomic DNA of five different cancer cell lines were accurately quantified by using LC-MS/MS. The data support our hypothesis that, after being incorporated into DNA, 6-thioguanine instead of S6-methylthio-2'-deoxyguanosine plays the major role to exert the cytotoxic effects of thiopurines. In addition, another nucleotide metabolite, 6-thioguanosine triphosphate was extracted and quantified by LC-MS/MS.;In Chapter 5, a strategy, including SILAC, affinity purification and LC-MS/MS, was employed to identify nuclear proteins that are capable of binding to 6-thioguanine-containing duplex DNA. The outcome of the study will facilitate the exploration of other mechanisms involved in the cytotoxicity of the thiopurine drugs.