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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Analysis of Oligonucleotide Mixtures by Electrospray Ionization-Mass Spectrometry

Xueheng Cheng, David C. Gale, Harold R. Udseth and Richard D. Smith*
Chemical Sciences Department, Pacific Northwest Laboratory, Richland, WA 99352

Our aim is to develop electrospray ionization mass spectrometry (ESI-MS) methods for high speed DNA sequencing of oligonucleotide mixtures, that can be integrated into an effective overall sequencing strategy. ESI produces intact molecular ions from DNA fragments of different size and sequence with high efficiency [1]. Our aim is to determine mass spectrometric conditions that are compatible with biological sample preparation and that avoid problems due to dissociation, aggregation, or adduction of the ionized DNA fragments. Oligonucleotide ions are typically produced from ESI with a broad distribution of net charge states for each molecular species (i.e., (M-nH)n-, where n is a series of integers), and thus leading to difficulties in analysis of complex mixtures [1]. To make identification of each component in a sequencing mixture possible, the charge states of molecular ions can be reduced by manipulating the ESI process and/or by using gas-phase reactions. The charge-state reduction methods being examined include: (1) reactions with organic acids (in the solution to be electrosprayed, the ESI-MS interface or the gas phase); (2) the labeling of the oligonucleotides with a designed functional group for production of molecular ions of very low charge states; and (3) the shielding of potential charge sites on the oligonucleotide phosphate/phosphodiester groups with polyamines (and the subsequent gas-phase removal of the neutral amines). In initial studies two methods for charge state reduction of gas phase oligonucleotide negative ions have been tested: (1) the addition of acids to the oligonucleotide solution and (2) the formation of diamine adducts followed by dissociation in the interface region [2]. In the first method, the efficiency of charge state reduction depends on the pKa, the concentration and the nature of the acids. Acetic and formic acids were found to be better reagents than HCl, CF3CO2H and H3PO4. The second method has the advantage that the stability of oligonucleotides is not affected but requires the optimization of the interface dissociation conditions and the amounts of diamine added to the oligonucleotide solution. Both methods show promise for charge state reduction and results have been demonstrated for small oligonucleotides (e.g., pd(T)12 and d(AGCT) ) [2]. Substantial reduction in spectral complexity was also observed for a four-component mixture of oligonucleotides upon charge state reduction. Our aim is to provide a basis for the development of an overall approach to high speed sequencing so as to provide a basis for the subsequent step of prototyping a cost effective high-throughput instrument for broad application.

* Corresponding author.

[1] "New Developments in Biochemical Mass Spectrometry: Electrospray Ionization", R. D. Smith, J. A. Loo, C. G. Edmonds, C. J. Barinaga, and H. R. Udseth, Anal. Chem., 62, 882-889 (1990).
[2] "Charge State Reduction of Oligonucleotide Negative Ions from Electrospray Ionization", X. Cbeng, D. C. Gale, H. R. Udseth, and R. D. Smith, Anal. Chem., submitted.