Christine M. Nelson, Lin Zhu, Wei Tang and Lloyd M. Smith
Department of Chemistry, University of Wisconsin, Madison, WI 53705
The development of MALDI and its demonstrated performance with large proteins, up to 300,000 daltons in size, has generated substantial interest in utilizing this technique as a replacement for the gel electrophoretic separation step in the Sanger sequencing protocol. If successful, the main advantages of this method over traditional gel-based methods are that polyacrylamide gels are no longer necessary and that the time for separation, detection and data acquisition is substantially reduced.
In developing this strategy, early studies of oligonucleotides have elucidated issues inherent to the analysis of nucleic acids using MALDI. These studies have shown that fragmentation is an important issue and is responsible for some of the current limitations associated with the technique.
Results in our laboratory and in others demonstrate that fragmentation is dependent on both oligonucleotide sequence and matrix composition.[l] Proposed fragmentation pathways consist of nucleobase protonation inducing base loss followed by backbone cleavage at the 3' C-O bond on the corresponding deoxyribose. A thorough study recently published from our laboratory elucidates this fragmentation mechanism and explains reasons for observed differences in base composition.[2] Studies in our laboratory investigating positive ion formation of oligonucleotides also support this mechanism. Currently, we are investigating the relationship between fragmentation propensity and matrix properties. Proton affinities of five common MALDI matrices have been measured and compared to fragmentation probabilities.
In addition to studying the fragmentation mechanism, we are using our current understanding to explore nucleotide modifications that may enhance the stability of DNA in the MALDI technique. Modifications on both the nucleobase and deoxyribose ring are being investigated. Experimental and theoretical approaches are being employed for this study.
By deepening our understanding of the fundamental chemistry of DNA fragmentation, we hope to be able to develop the MALDI technique into a powerful robust and versatile methodology for nucleic acid analysis.
1. Parr, G.R.; Fitzgerald, M.C.; Smith, L.M.; Rapid Commun. Mass Spectrom. 7, 63 (1993).
2. Zhu, L.; Parr, G.R.; Fitzgerald, M.C.; Nelson, C.M.; Smith, L.M.; J. Amer. Chem. Soc. 117, 6048 (1995).