MoP-05


VERIFICATION OF OLIGONUCLEOTIDE SYNTHESIS BY ESI-MS AND MS/MS UTILISING A NOVEL SEQUENCING ALGORITHM

J. I. Langridge1, E. Kapp1, T. McKenna1, D. Stranz1, T. Brown2 and J. Langley2

1Micromass UK Ltd., Tudor Road, Altrincham, Cheshire, WA14 5RZ, UK
2Dept. of Chemistry, University of Southampton, Southampton, UK


Synthetic oligonucleotides are playing an increasing role as hybridization probes to clone and synthesize genes, as potential therapeutic agents using antisense technology and as primers for polymerase chain reactions (PCR) and DNA sequencing. Traditionally the techniques used for the verification and analysis of oligonucleotides include DNA sequencing, HPLC analysis and PAGE. These techniques are labour-intensive and unreliable when a modified base has been incorporated.

Negative ion electrospray has been used for the analysis of synthetic oligonucleotides providing accurate molecular weight information. The use of MS-MS using a triple quadrupole or hybrid quadrupole orthogonal acceleration time of flight (QTOF) instrument gives added specificity over single MS, producing a series of multiply charged product ions allowing structural determination.

The MS/MS spectrum of an oligonucleotide (eg. 8mer) contains 200-500 mass measured peaks. The derivation of a sequence from a spectrum is a formidable task, due to the complexity of the multiply charged product ion spectrum and inherent mass ambiguities. The sequence has to be derived from sequence specific ion series both from the 3'-end and the 5'-end. A novel sequencing algorithm has been developed which utilises the inherent complexity of the MS/MS spectrum to produce a ranked sequence list.

In this study we present data from a range of synthetic oligonucleotides (4-17mers) analysed by ESI-MS/MS and demonstrate the merits of an automated sequencing program.