Richard D. Smith*, David C. Muddiman, Xueheng Cheng, S. A. Hofstadler, J. E. Bruce
Chemical Sciences Department and Environmental and Molecular Sciences Laboratory, Pacific Northwest Laboratory, Richland, WA 99352
This project is aimed at the development of a totally new concept for high speed DNA sequencing based upon the analysis of single (i.e., individual) large DNA fragments using electrospray ionization (ESI) combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In our approach, large single-stranded DNA segments extending to as much as 25 kilobases (and possibly much larger), is first transferred to the gas phase using ESI. The multiply-charged molecular ions are trapped in the cell of an FTICR mass spectrometer, where one or more single ion(s) can be selected for analysis in which its mass-to-charge ratio (m/z) is measured both rapidly and non-destructively. Single ion detection is achievable due to the high charge state of the electrosprayed ions and the unique sensitivity of the FTICR mass spectrometer developed at Pacific Northwest Laboratory.
Our efforts under the first several years of this project have demonstrated the capability for the formation, extended trapping, isolation, and monitoring of sequential reactions of highly charged DNA molecular ions with molecular weights well into the megadalton range [1-5]. We have shown that large multiply-charged individual ions of both single and double-stranded DNA anions can also be efficiently trapped in the FTICR cell, and their mass-to-charge ratios measured with very high accuracy. Thus, it is feasible to quickly determine the mass of each lost unit as the DNA is subjected to rapid reactive degradation steps. Our aim is to now develop methods based upon the use of ion-molecule or photochemical processes that can promote a stepwise reactive degradation of gas-phase DNA anions. Successful development of one of these approaches could greatly reduce the cost and enhance the speed of DNA sequencing, potentially allowing for sequencing DNA segments of more than 25 kilobase in length, on a time scale of minutes with negligible error rates with the added potential for conducting many such measurements in parallel. The techniques being developed promise to lead to a host of new methods for DNA characterization, potentially extending to the size of much larger DNA restriction fragments (>500 kilobases).
[1] "Trapping Detection and Reaction of Very Large Single Molecular Ions by Mass Spectrometry," R. D. Smith, X. Cheng, J. E. Bruce, S.A. Hofstadler and G.A. Anderson, Nature, 369, 137-139 (1994).
[2] "Charge State Shifting of Individual Multiply-Charged Ions of Bovine Albumin Dimer and Molecular Weight Determination Using an Individual-Ion Approach," X. Cheng, R. Bakhtiar, S. Van Orden, and R. D. Smith, Anal. Chem., 66, 2084-2087 (1994).
[3] "Trapping, Detection, and Mass Measurement of Individual Ions in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer,: J.E. Bruce, X. Cheng, R. Bakhtiar, Q. Wu, S.A. Hofstadler, G.A. Anderson, and R.D. Smith, J. Amer. Chem. Soc., 116, 7839-7847 (1994).
[4] "Direct Charge Number and Molecular Weight Determination of Large Individual Ions by Electrospray Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry", R. Chen, Q. Wu, D.W. Mitchell, S.A. Hofstadler, A.L. Rockwood, and R. D. Smith, Anal. Chem., 66, 3964-3969 (1994).
[5] "Trapping, Detection and Mass Determination of Coliphage T4 DNA (1.1 x 108 Da) Ions by Electrospray Ionization Fourier Transform Ion Cyclotron Reasonance Mass Spectrometry" R. Chen, X. Cheng, D.W. Mitchell, S.A. Hofstadler, A.L. Rockwood, Q. Wu, M.G. Sherman and R.D. Smith, Anal. Chem., 67, 1159-1163 (1995).
*This work was supported through the U.S. Department of Energy. Pacific Northwest Laboratory is operated by Battelle Memorial Institute through Contract No. DE-AC06-76RLO 1830.