Depurination of DNA and matrix-assisted laser desorption/ionization mass spectrometry

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of DNA suffers from low sensitivity and a limited mass range, allowing only the analysis of DNA fragments with up to 80 nucleotides. Mass range limitation is caused by depurination and adduct formation. Here the mechanism of acidic depurination of DNA with diester and triester backbones was investigated. Previously, undetected intermediates of depurination were identified by freezing the depurination reaction in the MALDI sample preparation. The products detected allow conclusions about the mechanism of depurination in aqueous solution to be made. A different mechanism of depurination for adenine and guanine is suggested, namely that adenine is substituted and guanine yields a mixture of substitution and elimination products which indicates that only guanine yields an oxocarbenium ion. A didehydro-2-deoxyribose product is detected on deguanylation. These results demonstrate the usefulness of MALDI for mechanistic investigations. Small, completely debased oligonucleotides were also detected in positive ion mode when charge tag technology was applied. Insight into the ionization mechanism of DNA during MALDI is gained. Complete depurination of DNA, which might be a useful tool for sizing large DNA products by MALDI, does not seem feasible because a degree of backbone cleavage is observed under acidic depurination conditions.