Characterisation of product ions in high-energy tandem mass spectra of protonated oligonucleotides formed by electrospray ionisation

Tandem mass spectra of positively charged ions of a range of small oligonuc leotides (6-10 mers) have been examined in detail using a hybrid magnetic s ector time-of-flight mass spectrometer. This has a higher range of collisio n energies (up to E(lab)400 eV) and a different timescale of dissociation c ompared to triple quadrupole and quadrupole ion trap instruments. Under the se conditions loss of neutral guanine and cytosine bases is frequently obse rved and this results in additional product ions comprising strands of the phosphodiester backbone with only 1-3 bases (especially adenine and thymine ) attached. The relative abundance of ions from the bases (BH2+) and ions f rom loss of a neutral base with the chain remaining intact also do not foll ow a clear trend in contrast to previous studies on triple quadrupoles, exc ept that a protonated base ion derived from thymine is never observed. The relative abundance of the remaining BH2+ ions varied with both oligonucleot ide sequence and the charge on the precursor, suggesting that under the col lision conditions employed, the relative proton affinities of the bases ade nine, cytosine, and guanine are not the only factors influencing ion abunda nce. For two of the oligonucleotides, 5'-CACGTG-3' and 5'-CGTACG-3', all of the fragment ions were also generated via collisional activation in the so urce and their tandem mass spectra examined to assist in the identification of the product ions observed in the tandem mass spectra of the intact olig onucleotides. These data also provide new information concerning the mechan isms by which oligonucleotides fragment in the positive ion mode. For examp le, the ion at m/z 81, which has been alternatively attributed to either a phosphate ion or to a sugar ion, is shown here to be derived principally fr om the sugar. This suggests that the sugar residues rather than the phospha te backbone are the most likely site of protonation after the nucleobases. Finally, tandem mass spectra of selected [M + H](+) and [M + 2H](2+) precur sors have been compared to the tandem mass spectra of the corresponding [M - H](-) and [M - 2H](2-) ions obtained under the same collision conditions. In the case of singly charged precursors, the formation of singly charged sequence ions is equally constrained by the fact that loss of neutral thymi ne is strongly disfavoured and consequently the sequence ions (w(n) and a(n )-B-n) observed in each case are similar. In contrast, for the doubly charg ed precursors there are marked differences in the relative abundance of the se sequence ions because loss of thymine as an anion to yield singly charge d sequence ions is possible for the deprotonated [M - 2H](2-) precursors wh ereas the analogous pathway from the doubly protonated precursors is clearl y unfavourable. (Int J Mass Spectrom 194 (2000) 269-288) (C) 2000 Elsevier Science B.V.