Controlling DNA fragmentation in MALDI-MS by chemical modification

Fragmentation has proven to be a major factor limiting accessible mass range, sensitivity, and mass resolution in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous work has shown that this DNA fragmentation is strongly dependent on both the MALDI matrix and the nucleic acid sequence employed. Fragmentation is initiated by nucleobase protonation, leading to cleavage of the N-glycosidic bond with base loss, followed by cleavage of the phosphodiester backbone. In this study, asymmetric oligonucleotides incorporating cytidine and cytidine analogs such as 5-methyl-2'-deoxycytidine, 5-bromo-2'-deoxycytidine, aracytidine, and 2'-fluorodeoxycytidine nucleosides were used to systematically investigate the influence of the structural changes on the stability of the N-glycosidic bond. Modifications of the deoxyribose sugar ring by replacing the 2'-hydrogen with more electron-withdrawing groups such as the hydroxyl or fluoro group stabilize the N-glycosidic bond to a greater extent than the C5 nucleobase modifications, 2'-Hydroxyl and 2'-fluoro groups respectively are shown to partially or completely block fragmentation at the modified nucleosides. Mixtures of oligonucleotides incorporating such modifications demonstrate remarkably extended accessible mass range, as well as increased sensitivity and mass resolution. The stabilization provided by these chemical modifications also expands the range of matrices useful for nucleic acid analysis, yielding in some cases greatly improved performance.