Homogeneous Preparations of 3′-Phosphoglycolate-Terminated Oligodeoxynucleotides from Bleomycin-Treated DNA as Verified by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Abstract

Single- and double-strand breaks bearing 3′-phosphoglycolate termini are among the most frequent lesions formed in DNA by ionizing radiation and other oxidative mutagens. In order to obtain homogeneous preparations of defined 3′-phosphoglycolate substrates for repair studies, 5′- 32P-end-labeled partial duplex DNAs were treated with bleomycin, and individual cleavage products were isolated from polyacrylamide gels. The fragments were then treated with alkaline phosphatase and further purified by reverse-phase HPLC. Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry of the purified oligomers produced molecular ions of the expected masses, with no detectable contaminants. Gas-phase sequencing by tandem mass spectrometry of these single species yielded the expected sequence ions and confirmed the presence of phosphoglycolate on the 3′-terminal fragments only. The fragments could be relabeled with polynucleotide kinase to yield highly purified, high-specific-activity substrates for repair studies.