Skip to main content
Download PDF

Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

Journal of the American Society for Mass Spectrometry volume 13, pages 936–945 (2002)Cite this article

Abstract

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2′-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions.

References

  1. Moser, H. E.; Dervan, P. B. Sequence-Specific Cleavage of Double Helical DNA by Triple Helix Formation. Science 1987, 238, 645–650.

    CAS  Article  Google Scholar 

  2. Nordhoff, E.; Kirpekar, F.; Roepstorff, P. Mass Spectrometry of Nucleic Acids. Mass Spectrom. Rev. 1996, 15, 67–138.

    Article  Google Scholar 

  3. Limbach, P. A. Indirect Mass Spectrometric Methods for Characterizing and Sequencing Oligonucleotides. Mass Spectrom. Rev. 1996, 15, 297–336.

    CAS  Article  Google Scholar 

  4. Murray, K. K. DNA Sequencing by Mass Spectrometry. J. Mass Spectrom. 1996, 31, 1203–1215.

    CAS  Article  Google Scholar 

  5. Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Mass Spectrometry. Anal. Chem. 1998, 70, 647R-716R.

    CAS  Article  Google Scholar 

  6. McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides. J. Am. Soc. Mass Spectrom. 1992, 3, 60–70.

    CAS  Article  Google Scholar 

  7. McLuckey, S. A.; Habibi-Goudarzi, S. Ion Trap Tandem Mass Spectrometry Applied to Small Multiply Charged Oligonucleotides with a Modified Base. J. Am. Soc. Mass Spectrom. 1994, 5, 740–747.

    CAS  Article  Google Scholar 

  8. McLuckey, S. A.; Habibi-Goudarzi, S. Decomposition of Multiply Charged Oligonucleotide Anions. J. Am. Chem. Soc. 1993, 115, 12085–12096.

    CAS  Article  Google Scholar 

  9. Barry, J. P.; Vouros, P.; Van Schepdael, A.; Law, S.-J. Mass and Sequence Verification of Modified Oligonucleotides Using Electrospray Tandem Mass Spectrometry. J. Mass Spectrom. 1995, 30, 993–1006.

    CAS  Article  Google Scholar 

  10. Little, D. P.; Aaserud, D. J.; Valaskovic, G. A.; McLafferty, F. W. Sequence Information from 42–108 mer DNAs (Complete for a 50 mer) by Tandem Mass Spectrometry. J. Am. Chem. Soc. 1996, 118, 9352–9359.

    CAS  Article  Google Scholar 

  11. Marzilli, L. A.; Barry, J. A.; Sells, T.; Law, S.-J.; Vouros, P.; Harsch, A. Oligonucleotide Sequencing Using Guanine-Specific Methylation and Electrospray Ionization Ion Trap Mass Spectrometry. J. Mass Spectrom. 1999, 34, 276–280.

    CAS  Article  Google Scholar 

  12. Christian, N. P.; Reilly, J. P.; Mokler, V. R.; Wincott, F. E.; Ellington, A. D. Elucidation of the Initial Step of Oligonucleotide Fragmentation in Matrix-Assisted Laser Desorption/ Ionization Using Modified Nucleic Acids. J. Am. Soc. Mass Spectrom. 2001, 12, 744–753.

    CAS  Article  Google Scholar 

  13. Gross, J.; Leisner, A.; Hillenkamp, F.; Hahner, S.; Karas, M.; Schäfer, J.; Lützenkirchen, F.; Nordhoff, E. Investigations of the Metastable Decay of DNA under Ultraviolet Matrix-Assisted Laser Desorption/Ionization Conditions with Post-Source-Decay Analysis and Hydrogen/Deuterium Exchange. J. Am. Soc. Mass Spectrom. 1998, 9, 866–878.

    CAS  Article  Google Scholar 

  14. Little, D. P.; Chorusch, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. Rapid Sequencing of Oligonucleotides by High-Resolution Mass Spectrometry. J. Am. Chem. Soc. 1994, 116, 4893–4897.

    CAS  Article  Google Scholar 

  15. Cerny, R. L.; Tomer, K. B.; Gross, M. L.; Grotjahn, L. Fast Atom Bombardment Combined with Tandem Mass Spectrometry for Determining Structures of Small Oligonucleotides. Anal. Biochem. 1987, 165, 175–182.

    CAS  Article  Google Scholar 

  16. Rodgers, M. T.; Campbell, S.; Marzluff, E. M.; Beauchamp, J. L. Low-Energy Collision-Induced Dissociation of Deprotonated Dinucleotides: Determination of the Energetically Favored Dissociation Pathways and the Relative Acidities of the Nucleic Acid Bases. Int. J. Mass Spectrom. Ion Processes 1994, 137, 121–149.

    CAS  Article  Google Scholar 

  17. McLuckey, S. A.; Vaidyanathan, G.; Habibi-Goudarzi, S. Charged versus Neutral Nucleobase Loss from Multiply Charged Oligonucleotide Anions. J. Mass Spectrom. 1995, 30, 1222–1229.

    CAS  Article  Google Scholar 

  18. Wang, Z.; Wan, K. X.; Ramanathan, R.; Taylor, J. S.; Gross, M. L. Structure and Fragmentation Mechanisms of Isomeric T-Rich Oligodeoxynucleotides: A Comparison of Four Tandem Mass Spectrometric Methods. J. Am. Soc. Mass Spectrom. 1998, 9, 683–691.

    CAS  Article  Google Scholar 

  19. Wan, K. X.; Gross, M. L. Fragmentation Mechanisms of Oligodeoxynucleotides: Effects of Replacing Phosphates with Methylphosphonates and Thymines with Other Bases in T-rich Sequences. J. Am. Soc. Mass Spectrom. 2001, 12, 580–589.

    CAS  Article  Google Scholar 

  20. Panico, M.; Sindona, G.; Uccella, N. Bioorganic Applications of Mass Spectrometry. 3. Fast-Atom-Bombardment-Induced Zwitterionic Oligonucleotide Quasimolecular Ions Sequenced by MS/MS. J. Am. Chem. Soc. 1983, 105, 5607–5610.

    CAS  Article  Google Scholar 

  21. Gross, J.; Hillenkamp, F.; Wan, K. X.; Gross, M. L. Metastable Decay of Negatively Charged Oligodeoxynucleotides Analyzed With Ultraviolet Matrix-Assisted Laser Desorption/Ionization Post-Source Decay and Deuterium Exchange. J. Am. Soc. Mass Spectrom. 2001, 12, 180–192.

    CAS  Article  Google Scholar 

  22. Wan, K. X.; Gross, J.; Hillenkamp, F.; Gross, M. L. Fragmentation Mechanisms of Oligodeoxynucleotides Studied by H/D Exchange and Electrospray Ionization Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 193–205.

    CAS  Article  Google Scholar 

  23. Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McClosky, J. A. Interpretation of Oligonucleotide Mass Spectra for Determination of Sequence Using Electrospray Ionization and Tandem Mass Spectrometry. Anal. Chem. 1996, 68, 1989–1999.

    CAS  Article  Google Scholar 

  24. Tang, W.; Zhu, L.; Smith, L. M. Controlling DNA Fragmentation in MALDI-MS by Chemical Modification. Anal Chem. 1997, 69, 302–312.

    CAS  Article  Google Scholar 

  25. Griffey, R. H.; Greig, M. J.; An, H.; Sasmor, H.; Manalili, S. Targeted Site-Specific Gas-Phase Cleavage of Oligoribonucleotides. Application in Mass Spectrometry-Based Identification of Ligand Binding Sites. J. Am. Chem. Soc. 1999, 121, 474–475.

    CAS  Article  Google Scholar 

  26. Kirpekar, F.; Krogh, T. N. RNA Fragmentation Studied in a Matrix-Assisted Laser Desorption/Ionization Tandem Quadrupole/Orthogonal Time-of-Flight Mass Spectrometer. Rapid Commun. Mass Spectrom. 2001, 15, 8–14.

    CAS  Article  Google Scholar 

  27. Méndez-Bernal, E.; Leumann, C. J. Conformational Diversity versus Nucleic Acid Triplex Stability, a Combinatorial Study. J. Biol. Chem. 2001, 276, 35320–35327.

    Article  Google Scholar 

  28. Users Manual 56-1111-56: Gene Assembler Special/4 Primers; Amersham Pharmacia Biotech: Piscataway, NJ;.

  29. Greco, F.; Liguori, A.; Sindona, G.; Uccella, N. Gas-Phase Proton Affinity of Deoxyribonucleosides and Related Nucleo-bases by Fast Atom Bombardment Tandem Mass Spectrometry. J. Am. Chem. Soc. 1990, 112, 9092–9096.

    CAS  Article  Google Scholar 

  30. Liguori, A.; Napoli, A.; Sindona, G. Survey of the Proton Affinities of Adenine, Cytosine, Thymine and Uracil Dideoxyribonucleosides, Deoxyribonucleosides and Ribonucleosides. J. Mass Spectrom. 2000, 35, 139–144.

    CAS  Article  Google Scholar 

  31. Clauwaert, J.; Stockx, J. Interactions of Polynucleotides and Their Components: I. Dissociation Constants of the Bases and Their Derivatives. Z. Naturforsch. B 1968, 23, 25–30.

    CAS  Google Scholar 

  32. Bartlett, M. G.; McCloskey, J. A.; Manalili, S.; Griffey, R. H. The Effect of Backbone Charge on the Collision-Induced Dissociation of Oligonucleotides. J. Mass Spectrom. 1996, 31, 1277–1283.

    CAS  Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland

    Stefan Schürch, Eloy Bernal-Méndez & Christian J. Leumann

Authors
  1. Stefan Schürch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eloy Bernal-Méndez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian J. Leumann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Schürch.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Schürch, S., Bernal-Méndez, E. & Leumann, C.J. Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides. J Am Soc Spectrom 13, 936–945 (2002). https://doi.org/10.1016/S1044-0305(02)00413-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1016/S1044-0305(02)00413-0

Keywords

  • Backbone Fragmentation
  • Backbone Cleavage
  • Base Loss
  • High Proton Affinity
  • Electrospray Tandem Mass Spectrometry