DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

36. DNA Characterization by Electrospray Ionization-FTICR Mass Spectrometry

David S. Wunschel, Bingbing Feng, Ljiljana Pasa Tolic, Mary S. Lipton, and Richard D. Smith

Pacific Northwest National Laboratory, Richland, WA 99352

rd_smith@pnl.gov

Mass spectrometry offers the potential for high speed DNA sequencing and ultra-sensitive characterization. Ongoing work in the laboratory is exploring approaches based upon electrospray ionization (ESI) and/or Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. These efforts have included advanced methods for the characterization of polymerase chain reaction (PCR) products¹, enzymatically produced oligonucleotide mixtures, modified DNA and the development of methods for the analysis of DNA large fragments. High mass accuracy measurements for PCR products allowing a single base substitutions to be detected at >250 bp level with de novo identification of an unreported base substitution. This capability also allows the identification of small differences in mass such as those arising from methylation². Study of DNA damage/modifications in their sequence context will likely have to occur from within multi-component mixtures. The capability for this has been demonstrated using a multi-component reaction where a base pair deletion was identified with the putative identification of inter-operon variability within a single bacterial strain. These efforts are also being extended to exploit the non-destructive nature of FTICR for recovery (i.e., "soft-landing") of mass-selected modified DNA segments, following high resolution FTICR analysis and separation (i.e., high resolution sorting), for subsequent cloning or PCR³. This capability allows for the direct selection and analysis of individual components from within mixtures. Alternatively, DNA species that cannot be identified through traditional sequencing methodologies, those containing base modifications, can be isolated and the nature and position of the modification identified. Most importantly this provides an approach for identification of low abundance modifications where few if any alternatives for their detection exist.

• D. S. Wunschel, D. C. Muddiman and R. D. Smith, Advances in Mass Spectrometry, Volume 14, E.J. Karjalainen, A.E. Hesso, J.E. Jalonen, U.P. Karjalainen, Eds., Elsevier Science Publishers B.V., Amsterdam, 377-406 (1998).
• D. S. Wunschel, L. Pasa Tolic, B. Feng and R. D. Smith, J. Amer. Soc. Mass Spectrom., in press.
• B. Feng, D. S. Wunschel, C. D. Masselon, L. Pasa-Tolic and R. D. Smith, J. Amer. Chem Soc., 121, 8961-8962 (1999).