Sequence analysis of oligodeoxyribonucleotides by mass spectrometry. 1. Dinucleoside monophosphates

Abstract

The base components of underivatized oligodeoxynucleotides can be determined qualitatively by mass-spectral analysis at the nanogram level. The thermal and electron-impact conditions of the spectrometer allow the cleavage of the phosphodiester bonds of the oligonucleotide chain, resulting in fragments (I), whose mass identifies the base, and other fragments (II), which contain the purine or pyrimidine base plus portions of the deoxyribose and the phosphate moieties. A study of 16 dinucleoside monophosphates indicates that the relative intensities of the m/e values of the type II fragments are significantly and reproducibly different for sequence isomers. From the complex spectra of dinucleoside monophosphates, specific ions for each mono-nucleotide residue have been selected which reveal the location (5' or 3' terminus) of the bases in the dinucleoside monophosphate. These ions appear in spectra of deoxyribonucleic acid fragments as well as in model compounds. A simple computer program has been devised which utilizes ion ratio values to determine sequence. The method is applicable to oligonucleotides of longer length.

Direct analysis in real time (DART) mass spectrometry of nucleotides and nucleosides: elucidation of a novel fragment [C5H5O]+and its in-source adducts

“…Evidence to support this conclusion comes from the observation that the corresponding peak pattern with an increment of 80 m/z units is not detected in the case of dTMP, which does not contain exocyclic amino groups on its nucleobase. Our observation is also consistent with a previous report that unlike other nucleobases, thymine and uracil could not form adducts with a PO 3 moiety because of the lack of an exocyclic nitrogen [26]. Interestingly, the addition reactions between several equivalents of 1 and two nucleobases (adenine and cytosine) were also detected.…”

“…However, this conclusion does not conflict with our proposed mechanisms because elimination reactions are commonly observed under pyrolytic conditions [23]. Wiebers and Shapiro reported elimination reactions at the 5=-and 3=-positions of nucleotides under pyrolytic conditions using electron-ionization (EI) mass spectrometry [26]. Such eliminations were also observed in the fragmentation analysis of DNA using pyrolysis field desorption mass spectrometry [27].…”

“…In our literature search for pyrolysis of DNA (mostly published in the 1970s and the 1980s), we found out that very few groups reported the observation of the furantype fragments with m/z 81 partially due to the mass range used (above m/z 100) [26,29]. In addition, the proposed structures for this fragment in the published papers were somewhat contradictory.…”

Identification of carbohydrate and nucleic acid biomarkers in the pyrolysis electron ionization-high-resolution mass spectrum of Brucella neotomae

“…Each nucleotide is composed of three separate units: a five carbon sugar, either ribose (in RNA) or deoxyribose (in DNA), a nitrogen base and a molecule of phosphate, PO 4 3À . Nucleic acids have been studied by several mass spectrometric (MS) techniques including electrospray (ESI-MS) [17][18][19][20], matrix-assisted laser desorption/ionization (MALDI-MS) [20][21][22][23][24], EI-MS and Py-MS [25][26][27][28][29]. Primary fragmentation of any polydeoxyribonucleotide upon pyrolysis involves cleavage at the phosphodiester bonds linking the nucleotide residues.…”

“…Primary fragmentation of any polydeoxyribonucleotide upon pyrolysis involves cleavage at the phosphodiester bonds linking the nucleotide residues. Subsequent EI+ fragmentation result in ions which are diagnostic for the common nucleotide components of the polynucleotide [27][28][29][30][31], including intact bases or even more or less severely dehydrated nucleosides.…”