Sequence analysis of oligodeoxyribonucleotides by mass spectrometry

Low-resolution mass spectra have been obtained on the following general types of mono-, di-, tetra,- and hexa-deoxyribonucleotides: pX, Xp, XpY, pXpY, pXp, pXpYp, pXpYpXpY and on trifluoroacetylated XpY, YpX, pXpYpXpY, XpYpXpY, and XpYpXpYpXpY (where X and Y indicate the purine and pyrimidine nucleosides, cytidine, thymidine, adenosine, or guanosine). The mass spectral fragmentation data show that: 1. (a) The purine and pyrimidine components of deoxyribonucleotides and oligodeoxyribonucleotides can be readily identified from underivatized material. 2. (b) Ions containing the nucleoside moiety plus portions of the phosphate moiety were not found; consequently, sequence information cannot be derived from the position of the phosphate moiety. 3. (c) Underivatized sequence isomers of deoxyribo- dinucleoside monophosphates cannot be distinguished; however, trifluoroacetylation of the molecules yields diagnostic ions which allow sequence determination. 4. (d) Oligodeoxyribonucleotides can be derivatized to yield ions which are diagnostic for the 3′- and 5′-terminals of the molecule and ions which identify all the purine and pyrimidine bases present in the interior of the molecule. The sequence of the internal residues, as well as the identity of the terminal nucleosides, can be determined by the application of the technique to the isolated products of limited venom phosphodiesterase digestion of the oligodeoxyribonucleotide, which are oligonucleotides of decreasing chain length. The identification of the 3′-terminal nucleoside of each oligonucleotide allows the reconstruction of the parent molecule. The simplicity of the derivatization and analysis and the extreme sensitivity of the method indicate the potential usefulness of the method.