Mass and sequence verification of modified oligonucleotides using electrospray tandem mass spectrometry

Electrospray coupled with tandem mass spectrometry is shown to have utility in determining the molecular mass and the sequence of short modified oligonucleotides. The product ion spectrum of a triply deprotonated molecule precursor is dominated by ions formed via two fragmentation pathways: first, fragmentation across the 5′PO phosphate bond, and second, loss of a non-terminal base with concomitant cleavage of the ribose 3′CO bond. If either of these two pathways is complete with fragmentation occurring at each equivalent position along the phosphodiester backbone, then the oligomer may be sequenced. Alternatively, if both these pathways are only partially complete, their complementarity may be used for more extensive sequencing. The inductive effect of the modification on the nucleobase is shown to influence the latter fragmentation process. The greater the electron affinity of the base modification, the more facile is the loss of that base when the molecule is collisionally activated. In fact, this latter fragmentation dominates the product ion spectrum when a nucleobase contains a substituent that is highly inductively withdrawing. This domination can be such that few other first-generation fragment ions are present in the product ion spectrum. Sequence information may then require utilization of second-generation product ions.