Comparative sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry.
An algorithm was developed for the computer-aided interpretation of fragment ion spectra from collision-induced dissociation of multiply charged oligodeoxynucleotide ions generated by electrospray ionization. The method compares the experimental spectrum to the m/z values predicted by employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between measured spectrum and predicted m/z values. To substantiate the identity of investigated sequence and reference sequence, or to identify point mutations or insertions/deletions, the reference sequence is systematically varied by incorporating all four possible nucleotides A, T, G, and C at each position in the sequence followed by identification of the correct sequence by the lowest fitness value. Collision energy was shown to have a major impact on the interpretability of the tandem mass spectra by the comparative sequencing algorithm, and the optimal collision energy depended on the length of the fragmented oligodeoxynucleotide. The analytical system was successfully applied to verify DNA sequences as well as to detect and localize point mutations or insertions/deletions in 5-51-mer oligodeoxynucleotides.
Oberacher
Herbert
H
Institute of Analytical Chemistry and Radiochemistry and Institute of General, Inorganic, and Theoretical Chemistry, Leopold-Franzens-University, Innsbruck, Austria.
Wellenzohn
Bernd
B
Huber
Christian G
CG
D000465:Algorithms; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
D009838:Oligodeoxyribonucleotides