Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

Abstract

We present the first global computer-aided sequencing algorithm for the de novo determination of short nucleic acid sequences. The method compares the fragment ion spectra generated by collision-induced dissociation of multiply charged oligodeoxynucleotide-ions to the m/z values predicted 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 the measured spectrum and predicted m/z values. In order to find the sequence most closely matching the experimental spectrum, starting from a given nucleotide composition all possible oligonucleotide sequences are assembled followed by identification of the correct sequence by the lowest fitness value. Using this concept, sequences of 5-to 12-mer oligodeoxynucleotides were successfully de novo determined. High sequence coverage with fragment ions was essential for obtaining unequivocal sequencing results. Moreover, the collision energy was shown to have an impact on the interpretability of tandem mass spectra by the de novo sequencing algorithm. Experiments revealed that the optimal collision energy should be set to a value just sufficient for complete fragmentation of the precursor ion. T he hyphenation of liquid chromatography (LC) to mass spectrometry (MS) is one of the most powerful methods for the characterization of oligonucleotides today [1]. In this context the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ICEMS) has emerged as a versatile tool for the analysis of single-and double-stranded nucleic acids ranging in size from a few nucleotides (nt) up to several hundred base pairs (bp) [2][3][4]. Although the occurrence of a sequence variation is predictable on the basis of molecular mass measurements [5][6][7], detailed sequencing information can only be obtained from selective decomposition of oligonucleotides by tandem mass spectrometry (MSMS).The principles of gas-phase collision-induced dissociation (CID) reactions of oligonucleotides have been studied extensively [8 -17] and the resulting product ion spectra are predictable on the basis of known fragmentation pathways. Consequently, MSMS of oligodeoxynucleotides has been successfully applied to characterize or confirm structures [11], to detect chemical modifications [2,10,18], and to identify sequence variations [14, 19 -21]. However, the deduction of sequence information is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS. Hence, automation of pro...

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