BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. zyxwv 16, 451454 (1988) zyxw Sequence Effect on the Slow Degradations of Dinucleotides by Fast Atom Bombardment Tandem Mass Spectrometry' Angelo Liguori, Giovanni Sindona and Nicola Uccella Dipartimento di Chimica, Universita della Calabria, 1-87030 Arcavacata di Rende, Italy The mass-analysed ion kinetic energy spectrum of isomeric dinucleotides having dTpdU and dUpdT structure is characterized by the preferred elimination zyxwvu of the pyrimidine base linked to the 5' end deoxyribose unit. Similar results have zyxwvuts been obtained with differently substituted dimers such as dCpdT and DMTdTdS)dU. The observed selectivity in the glycosidic bond dissociation provides a straightforwardcriterion for the sequencing of the sampled species. INTRODUCTION The introduction of fast atom bombardment (FAB) mass spectrometry' in the structure determination of nucleic acid oligomers obtained by chemical synthesis dates back to the early applications of the method- 010gy.~ It was recognized that it was possible to obtain structuraI information on isomeric 3'-5' and 5'-5' dithy- midilic acids from the degradations of their [M zyxwvu - HI- species. Longer strands of 3'-5' linked oligonucleotides are released into the gas phase as monocharged species, where n - 1 protonated nucleobases balance the n negative charges placed on the diesterophosphate units : this structural feature affects their unimolecular and collision-induced dissociations (CID).4 The applicability of FAB mass spectrometry to the characterization of DNA segments of unknown struc- ture requires the identification of those spectral para- meters which allow the unambiguous determination of the sequence of the sampled species. The criterion based on the ratio of the fragments formed by competitive internucleotidic bond breakage from the 3' or 5' ends' is not generally ~perative.~ Many factors affect this process, including the chemical environment on the reacting centre and the sequence of the oligomer, as pre- viously shown for partially protected oligomers,6 and the matrix effect.' The latter has been verified with fully protected nucleotides which undergo selective solvolysis of the triesterophosphate moiety driven by bimolecular processes which are known to occur in the condensed layers during an FAB experiment.'-'' The tandem mass spectrometry (MS/MS) methodology" has therefore been applied to the identification of those reaction channels which, eventually, could allow a straightfor- ward structural assignment of isomeric dinucleotides. EXPERIMENTAL The dinucleotides 14 have been synthesized according to published procedures" and purified by thin-layer chromatography (TLC).13 The mass spectra have been obtained on a Vacuum Generators (VG) ZAB-2F instrument, operated at an accelerating potential of 8 keV, by using the M-SCAN steerable FAB gun. A neu- tral Xe beam of 9.5 keV energy and a neutral current of approximately 10 pA was employed. 2 pl of water solu- tions of the ammonium salts of 1-4 were mixed with 2 pl of glycerol directly on the FAB target. The spectra were recorded at loo0 resolution by scanning the mag- netic sector. Mass-analysed ion kinetic energy (MIKE) spectra were obtained by scanning upwards the electro- static sector at an accelerating potential of 8 keV. CID spectra were obtained by admitting air into the second field-free region collision cell, lowering to one-third the initial intensity of the main beam. RESULTS AND DISCUSSION ~~ Isomeric dTpdU (1) and dUpdT (2) dinucleotides bear nucleobases which differ only in the presence of a methyl group at position 5 of the pyrimidine moieties. The uracil and thymine residues should exert similar electronic effects both on the stability of the glycosidic bond and on the reactivity of the phosphate linkage. It should be expected, therefore, that the reaction paths populated by 1 and 2 in the gas phase would reflect the chemistry of a dinucleotide framework. The upper part of the FAB spectrum of the negative ions desorbed from glycerol/water solutions of both dinucleotides 1 and 2 was characterized by the presence of the fragments at zyxw m/z 307 and 321 which correspond to uracil (UMP) and thymine (TMP) monophosphates respectively. As can be easily detected from the spectra reported in Fig. 1, there is always a preference for the formation of m/z 321 which, however, corresponds to isomeric 5'-TMP and 3'-TMP originating from 1 and 2, respectively. This behaviour cannot be easily rational- ized in any case, whatever the mechanism; the informa- tion thus obtained cannot be used to distinguish among T-U and U-T sequences. On the contrary, the loss of 0887-61 34/88/24045144 zyxwvutsrqpo $05.00 zyxwvutsrq 0 1988 by John Wiley & Sons, Ltd.