GAS-CHROMATOGRAPHY MASS-SPECTROMETRY OF FREE RADICAL-INDUCED PRODUCTS OF PYRIMIDINES AND PURINES IN DNA

This chapter describes the use of the technique of gas chromatography-mass spectrometry (GC/MS) for chemical characterization and quantitative measurement of free radical-induced products of pyrimidines and purines in Deoxyribo-nucleic acid (DNA) and DNA-protein cross-links in nucleoprotein. Prior to analysis by GC/MS, DNA and nucleoprotein must be hydrolyzed. Acidic hydrolysis cleaves the glycosidic bonds between bases and sugar moieties in DNA and thus frees intact and modified bases. Enzymatic hydrolysis is used to hydrolyze DNA to nucleosides. In the case of DNA-protein cross-links, the simplest way for hydrolysis of nucleoprotein appears to be the standard method of protein hydrolysis—that is, hydrolysis with 6 M HCl, which cleaves peptide bonds in proteins as well as glycosidic bonds in DNA to free base-amino acid cross-links. Prior to hydrolysis, DNA and nucleoprotein samples should be extensively dialyzed against water and subsequently lyophilized. The GC/MS technique is applicable only to compounds that are volatile, or can be made sufficiently volatile. Bases, nucleosides, and DNA base-amino acid cross-links are not sufficiently volatile for gas chromatography, and thus must be converted into volatile derivatives. Generally, trimethylsilylation is the most widely used mode of derivatization. © 1990, Elsevier Inc. All rights reserved.