Computational and enzymatic digestion methods for the mass spectrometric analysis of oligonucleotide adducts

Permanent URL: http://hdl.handle.net/2047/d10016880
Title:
Computational and enzymatic digestion methods for the mass spectrometric analysis of oligonucleotide adducts
Creator:
Liao, Qing (Author)
Contributor:
Vouros, Paul (Advisor)
Mabrouk, Patricia Ann (Committee member)
Davies, Geoffrey (Committee member)
Forsyth, David A. (Committee member)
Publisher:
Boston, Massachusetts : Northeastern University, 2008
Date Accepted:
December 2008
Date Awarded:
May 2009
Type of resource:
Text
Genre:
Dissertations
Format:
electronic
Digital origin:
born digital
Abstract/Description:
The assignment of the sites of modification by different carcinogens within a target sequence is a very important topic in cancer etiology and a critical part in the analysis of DNA adducts is to develop a method to determine the site and frequency of modification on the DNA strands in order to better understand the relationship between the chemical behavior of different carcinogens and mutation hot spots. The initial focus was on the analysis of DNA adducts as "monomeric units", i.e., nucleosides or nucleotides, in part because they presented an easier target. In order to accomplish the goal of developing a sound methodology in correlating the adducts formation and gene sequence, it is necessary to have a fast and accurate methodology for structural characterization of oligonucleotide-carcinogen adducts from in vitro and in vivo sources using on-line separation coupled with MS or tandem MS. It is with the above considerations in mind that the theme of the dissertation was evolved. The aim of this dissertation's research has been to develop HPLC-MS methodology for the analysis of DNA adducts in the form of oligonucleotides. To achieve this goal, a thorough literature review was conducted first and a systematic approach with carefully designed experiments was carried out. The results presented in chapter 2 and chapter 3 have been published. In chapter 1, we review the highlights of mass spectrometric applications to the analysis of biomarkers indicative of DNA damage. We first present an almost historical summary of key advances in the field of DNA biomarkers in order to provide a better perspective of the problems and challenges associated with the problem. Beginning with a discussion of the early approaches, we review the basic strategies to DNA adduct analysis by mass spectrometry and then the most recent advances in the MS analysis of oligonucleotide adducts. Chapter 2 focuses on the development of DNA enzymatic digestion procedures that would cut DNA into longer fragments, providing more complete information about neighboring base effects on adduction. We present the development of an ion-pairing HPLC-MS method that has sufficient separation power, selectivity and sensitivity to investigate the enzymatic behavior of benzonase/alkaline phosphatase for the digestion of oligonucleotides and DNA. We demonstrate that benzonase/alkaline phosphatase is a promising choice for DNA and DNA-adduct related studies that require a non-specific enzyme. In this stage of our research a computer software program named GenoMass took its primary shape as the needs arose in automating the processing of mass spectral data, which was very critical in developing the methodology (Liao, Vouros et al. Anal. Chem. 2007, 79, 1907-1917). Chapter 3 focuses on the development of a novel computer software, for which we have coined the name GenoMass, to handle the myriad of data produced from the LC-MS analysis of DNA digests, since the generation of progressively longer oligonucleotides is likely to yield a multitude of isomeric species. Here, we describe a "reversed pseudo-combinatorial" approach for fragment identification and the software implementation of this approach. Combinatorial isomer libraries are generated in silico to represent the digestion products of oligonucleotides, DNA or DNA adducts of various sizes. The software automatically calculates ion masses of each isomeric segment of the library, searches for them in complicated LC-MS data, lists their intensities and plots extracted ion chromatograms (EIC). This customized new data analysis tool has enabled a study of the enzymatic behavior of a nuclease system in the digestion of normal and adducted DNA, and in the recognition of oligomers containing a carcinogen bound to a nucleobase. The software program potentially can be further expanded to postulate unknown DNA sequences and recognize the adduction sites. (Liao, Shen and Vouros, J. Mass Spectrom., in press, 2008). Chapter 4 explores the utility of the aforementioned procedures to examine the relationship between chemoselectivity and mutation in model oligonucleotides bearing a mutation hotspot to a specific carcinogen. The relationship between the preferential binding of the carcinogen N-acetoxy-2-acetylaminofluorene (AAAF) to oligonucleotides and the mutational hotspot was investigated using specially designed model oligonucleotides containing the NarI gene sequence, a known mutation target for AAAF. The measured molecular weights (MW) of adducted oligonucleotide tetramers were matched with those in the GenoMass generated database from the LC-ESI/MS analysis of the digests. The selectivity of adduct binding was evaluated semi-quantitatively by comparing relative abundances of the found adduct ions. This methodology was also extended to investigate trends in the preferential binding of AAF (N-acetyl-2-aminofluorene) on calf thymus DNA (ctDNA). Chapter 5 presents the future prospects of the research which will likely be pursued further based on the studies presented in this dissertation.
Subjects and keywords:
Chemistry
Oligonucleotide DNA adducts
DNA enzymatic digestion
GenoMass software
Ion-Pairing LC-MS
Oligonucleotides - Analysis
DNA adducts
Biochemical markers
Biochemistry
DOI:
https://doi.org/10.17760/d10016880
Permanent Link:
http://hdl.handle.net/2047/d10016880
Use and reproduction:
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