Analysis of M1G-dR in DNA by aldehyde reactive probe labeling and liquid chromatography tandem mass spectrometry.

A novel method for the measurement of pyrimido[1,2-a]purin-10(3H)one (M1G) has been developed. Previous methods for analysis of M1G have been confounded by the fact that this lesion exists in equilibrium between a ring-closed form and a ring-opened aldehyde form. Poor detection sensitivity of the aldehydic form can result from loss of the adduct during analysis by its reaction with amines or proteins. We utilized the aldehyde reactive probe (ARP) to produce a stable ARP-M1G-deoxyribose (ARP-M1G-dR) conjugate to minimize adduct loss. This conjugate has increased the hydrophobicity that enhances separation of ARP-M1G-dR from unmodified DNA nucleosides by using solid phase extraction. In addition, measuring ARP-M1G-dR by selective reaction monitoring (SRM) of the [ARP-M1G-dR + H]+ (635) --> [M1G + H]+ (188) transition increases the detection sensitivity by nearly an order of magnitude relative to the measurement of M1G-dR by SRM. For accurate measurement, analytical standard (AS) DNA and internal standard (IS) DNA were used. High purity 15N-labeled DNA was isolated from Escherichia coli that had been grown in minimum salt medium containing (15NH4)2SO4. The 15N-DNA and calf thymus DNA were treated with malondialdehyde to induce a high number of M1G adducts to prepare the IS and AS DNA, respectively. A consistent calibration curve was established from the analysis of 200 microg of blank DNA, 23 ng of IS DNA (400 fmol of 15N5-M1G-dR), and AS DNA containing 0-810 fmol of M1G-dR. With the use of this novel IS DNA and selective labeling, this assay is a useful tool for monitoring oxidative stress-induced DNA damage from small amounts of DNA without the need of a specific antibody or laborious procedures. By this assay, two M1G adducts/10(8) guanines can readily be detected. Furthermore, this approach should be applicable to the analysis of other aldehydic DNA adducts as well as the measurement of an array of DNA lesions.