THE EFFECT OF BACKBONE CHARGE ON THE COLLISION-INDUCED DISSOCIATION OF OLIGONUCLEOTIDES

Knowledge of the effects of structural changes in oligonucleotides on their dissociation reactions is important in the application of mass s pectrometry to sequence determination, The effect of backbone charge o n the collision-induced dissociation of multiply-charged oligonucleoti des produced by electrospray was explored by examination of models in which the normal phosphodiester linkage was partially replaced with an uncharged methylphosphonate (MP) linkage, Three different MP-containi ng oligonucleotides were studied, designed to represent a concentratio n of charge on the 5'- and 3'-ends of the molecule and with an even di stribution of charge along the backbone, compared with a control molec ule containing only phosphodiester linkages, In all MP-containing olig onucleotides charging of over 90% of phosphate groups were observed, c ompared with typical charging patterns of about 60% in normal all-phos phodiester oligonucleotides, This unexpected effect is attributed to c harge stabilization by interactions of charged sites with uncharged re sidues, Analysis of the collision-induced dissociation mass spectra sh owed that backbone cleavage occurred at every residue (w and a - base ion series), producing a full set of sequencing ions whether or not th e linkage at that site was formally charged, It is concluded that unde r the multiple collision conditions of the quadrupole collision cell t hat backbone cleavage proceeds through two generic pathways, one invol ving base loss followed by cleavage of the adjacent C3' - CO bond and the other requiring neither base loss nor charged phosphate at the cle avage site, These results suggest that backbone cleavage reactions in conventional phosphodiester oligonucleotides can occur at non-ionized linkage sites, of which there are a high proportion in both electrospr ay- and MALDI-produced molecular ions.