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Mass Spectrometry-Based Studies of DNA Lesions and Epigenetic Biomarkers

  • Author(s): Yu, Yang
  • Advisor(s): Wang, Yinsheng
  • et al.
Abstract

LC-MS has been employed as a powerful tool for elucidating structures of compounds as well as providing accurate and unambiguous quantification of analytes. The focus of this dissertation is placed on the development of analytical methods for reliable quantification of chemically modified nucleosides in DNA, including oxidative stress-induced DNA lesions, carboxymethylated DNA adducts, and DNA epigenetic marks.

In chapter 2, I employed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (NanoLC-NSI-MS2) coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. The quantification data suggested that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation.

In chapter 3, I developed a highly sensitive nLC-nESI-MS3 method for the simultaneous quantification of N6-CMdA, O6-CMdG and O6-MedG. I was able to measure the levels of these three lesions with the use of low-microgram quantities of DNA from cultured human skin fibroblasts and human colorectal carcinoma cells treated with azaserine, a DNA carboxymethylating agent. The method reported here will be useful for the future investigations about the repair of the carboxymethylated DNA lesions and about the implications of these lesions in carcinogenesis.

In chapter 4, I employed LC-MS3 and quantified the levels of 5-hmdC in different cancer cells treated with a glycolysis inhibitor or activators of AMP-activated protein kinase (AMPK) to gain insights into the relationship between aberrant metabolism and the levels of 5-hmdC in cancer cells. The data indicated that the altered metabolism in cancer cells could contribute, in part, to the diminished levels of 5-hmdC observed in cancer cells.

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