Fragmentation mechanisms of oligodeoxynucleotides: effects of replacing phosphates with methylphosphonates and thymines with other bases in T-rich sequences

This article reports another step in an ongoing effort to understand the fragmentation of T-rich oligodeoxynucleotides. We extended an earlier investigation of T-rich 4-mers [1] to T-rich 6-mers, 8-mers and 10-mers by using four different tandem mass spectrometric methods. The methods include low-energy collisionally activated decomposition (CAD) of electrospray ionization (ESI)-produced ions, source-CAD of ESI-produced ions, post-source decay (PSD), and CAD of matrix assisted laser desorption ionization (MALDI)-generated ions. The most abundant fragment ions produced from [M - 2H]^2- precursors upon low-energy CAD in an ion trap are the [a - B]^- and their complementary w ions. The predominant cleavage sites for T-rich oligodeoxynucleotides are always the 3′ C=O bonds adjoining a non-T nucleobase (i.e., a base with a higher proton affinity (PA) than that of T). The relative abundance of [a - B]^- correlates with the PAs of the nucleobases, underscoring the importance of proton transfer to the base. The propensity to form [a - B]^- ions falls in the order of G > C ≈ A ≫ T. Structural isomers up to 10-mers can be readily sequenced and distinguished with each of the four tandem mass spectrometric methods applied. The fragmentation of oligodeoxynucleotides in which various phosphates were replaced with methylphosphonate is a measure of the participation of the phosphate proton in the formation of [a - B]^- ions. For 4 and 5-mers, transfer of an acidic proton from the 5′-phosphate to the departing base is the initiating step in the formation of [a - B]^- ions.