Analysis of Oligonucleotides Using Capillary Zone Electrophoresis and Electrospray Mass Spectrometry

  • An Willems
  • Dieter L. Deforce
  • Jan Van Bocxlaer
Part of the Methods In Molecular Biology™ book series (MIMB, volume 384)


This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry for the single-step desalting, and separation, as well as characterization of oligonucleotides in the framework of quality control after synthesis. Separation is performed using a 25 mM ammonium carbonate buffer supplemented with 0.2 mM trans-1,2-diaminocyclohexane-N, N, N′, N′ id (CDTA) (pH 9.7). During the electrophoretic process, sodium and potassium ions are removed from the polyanionic backbone of the oligonucleotides by exchange of these ions with ammonium ions or by chelation on CDTA, thus eliminating a sample preparation step. A sample stacking procedure used to concentrate the samples on the CZE capillary is described. After analysis, the obtained spectrum is deconvoluted to the zero charge spectrum to yield the molecular mass of the oligonucleotide. A misincorporation of one nucleotide can be detected by a difference in mass.


Capillary zone electrophoresis electrospray mass spectrometry quadrupole time-of-flight mass spectrometer oligonucleotides quality control 



We thank Ing. S. Vande Casteele for all her help with the analysis of the samples. This work was supported by grant GOA99-120501-99 (Bijzonder OnderzoeksFonds Universiteit Gent).


  1. 1.
    De Bellis, G., Salani, G. (1997) Oligonucleotide analysis by capillary zone electrophoresis at low pH. Anal. Chim. Acta 345, 1–4.CrossRefGoogle Scholar
  2. 2.
    Gaus, H. J., Owens, S. R., Winniman, M., Cooper, S., Cummins, L. L. (1997) On-line HPLC electrospray mass spectrometry of phosphorothioate oligonucleotide metabolites. Anal. Chem. 69, 313–319.CrossRefPubMedGoogle Scholar
  3. 3.
    Apffel, A., Chakel, J. A., Fisher, S., Lichtenwalter, K., Hancock, W. S. (1997) Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry. Anal. Chem. 69, 1320–1325.CrossRefGoogle Scholar
  4. 4.
    Deforce, D. L. D., Raymackers, J., Meheus, L., Van Wijnendaele, F., De Leenheer, A., Van den Eeckhout, E. G. (1998) Characterization of DNA oligonucleotides by coupling of capillary zone electrophoresis to electrospray ionization Q-TOF mass spectrometry. Anal. Chem. 70, 3060–3068.CrossRefGoogle Scholar
  5. 5.
    Mangano, M. F., Battaglia, C., Salani, G., Rossi Bernardi, L., De Bellis, G. (1999) Composition dependent separation of oligonucleotides by capillary electrophoresis in acidic buffers with application to the quality control of synthetic oligonucleotides. J. Chromatogr. A 848, 435–442.CrossRefGoogle Scholar
  6. 6.
    Pietta, P. G., Mangano, M. F., Battaglia, C., Salani, G., Rossi Bernardi, L., De Bellis, G. (1999) Application of capillary electrophoresis at low pH to oligonucleotides quality control. J. Chromatogr. A 853, 355–358.CrossRefPubMedGoogle Scholar
  7. 7.
    Fountain, K. J., Gilar, M., Gebler, J. C. (2003) Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 17, 646–653.CrossRefPubMedGoogle Scholar
  8. 8.
    Huber, C. G., Krajete, A. (1999) Analysis of nucleic acids by capillary ion-pair reversed-phase HPLC coupled to negative-ion electrospray ionization mass spectrometry. Anal. Chem. 71, 3730–3739.CrossRefGoogle Scholar
  9. 9.
    Nordhoff, E., Kirpekar, F., Roepstorff, P. (1996) Mass spectrometry of nucleic acids. Mass Spectrom. Rev. 15, 67–138.CrossRefGoogle Scholar
  10. 10.
    Doktycz, M. J., Hurst, G. B., Habibi-Goudarzi, S., McLuckey, S. A., Tang, K., Chen, C. H., Uziel, M., Jacobson, K. B., Woychik, R. P., Buchanan, M. V. (1995) Analysis of polymerase chain reaction-amplified DNA products by mass spectrometry using matrix-assisted laser desorption and electrospray: current status. Anal. Biochem. 230, 205–214.CrossRefPubMedGoogle Scholar
  11. 11.
    Walters, J. J., Fox, K. F., Fox, A. (2002) Mass spectrometry and tandem mass spectrometry, alone or after liquid chromatography, for analysis of polymerase chain reaction products in the detection of genomic variation. J. Chromatogr. B 782, 57–66.CrossRefGoogle Scholar
  12. 12.
    Huber, C. G., Oberacher, H. (2001) Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry. Mass Spectrom. Rev. 20, 310–343.CrossRefPubMedGoogle Scholar
  13. 13.
    Bleicher, K., Bayer, E. (1994) Analysis of oligonucleotides using coupled high-performance liquid chromatography-electrospray mass spectrometry. Chromatographia 39, 405–408.CrossRefGoogle Scholar
  14. 14.
    Janning, P., Schrader, W., Linscheid, M. (1994) A new mass spectrometric approach to detect modifications in DNA. Rapid Commun. Mass Spectrom. 8, 1035–1040.CrossRefPubMedGoogle Scholar
  15. 15.
    Schrader, W., Linscheid, M. (1995) Determination of styrene oxide adducts in DNA and DNA components. J. Chromatogr. A 717, 117–125.CrossRefPubMedGoogle Scholar
  16. 16.
    Schrader, W., Linscheid, M. (1997) Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry. Arch. Toxicol. 71, 588–595.CrossRefPubMedGoogle Scholar
  17. 17.
    Barry, J. P., Muth, J., Law, S.-J., Karger, B. L., Vouros, P. (1996) Analysis of modified oligonucleotides by capillary electrophoresis in a polyvinylpyrrolidone matrix coupled with electrospray mass spectrometry. J. Chromatogr. A 732, {159–166.}CrossRefGoogle Scholar
  18. 18.
    Harsch, A., Vouros, P. (1998) Interfacing of CE in a PVP matrix to ion trap mass spectrometry: analysis of isomeric and structurally related (N-acetylamino)fluorene-modified oligonucleotides. Anal. Chem. 70, 3021–3027.CrossRefPubMedGoogle Scholar
  19. 19.
    Freudemann, T., von Brocke, A., Bayer, E. (2001) On-line coupling of capillary gel electrophoresis with electrospray mass spectrometry for oligonucleotide analysis. Anal. Chem. 73, 2587–2593.CrossRefPubMedGoogle Scholar
  20. 20.
    von Brocke, A., Freudemann, T., Bayer, E. (2003) Performance of capillary gel electrophoretic analysis of oligonucleotides coupled on-line with electrospray mass spectrometry. J. Chromatogr. A 991, 129–141.CrossRefGoogle Scholar
  21. 21.
    Liu, C., Wu, Q., Harms, A. C., Smith, R. D. (1996) On-line microdialysis sample cleanup for electrospray ionization mass spectrometry of nucleic acid samples. Anal. Chem. 68, 3295–3299.CrossRefPubMedGoogle Scholar
  22. 22.
    Huber, C. G., Buchmeiser, M. R. (1998) On-line cation exchange for suppression of adduct formation in negative-ion electrospray mass spectrometry of nucleic acids. Anal. Chem. 70, 5288–5295.CrossRefPubMedGoogle Scholar
  23. 23.
    Griffey, R. H., Greig, M. J., Gaus, H. J., Liu, K., Monteith, D., Winniman, M., Cummins, L. L. (1997) Characterization of oligonucleotide metabolism in vivo via liquid chromatography/electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer. J. Mass Spectrom. 32, 305–313.CrossRefPubMedGoogle Scholar
  24. 24.
    Ragas, J. A., Simmons, T. A., Limbach, P. A. (2000) A comparative study on methods of optimal sample preparation for the analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry. Analyst 125, 575–581.CrossRefPubMedGoogle Scholar
  25. 25.
    Huber, C. G., Krajete, A. (2000) Comparison of direct infusion and on-line liquid chromatography/electrospray ionization mass spectrometry for the analysis of nucleic acids. J. Mass Spectrom. 35, 870–877.CrossRefPubMedGoogle Scholar
  26. 26.
    Limbach, P. A., Crain, P. F., McCloskey, J. A. (1995) Molecular mass measurement of intact ribonucleic acids via electrospray ionization quadrupole mass spectrometry. J. Am. Soc. Mass Spectrom. 6, 27–39.CrossRefGoogle Scholar
  27. 27.
    Wolf, S. M., Vouros, P. (1995) Incorporation of sample stacking techniques into the capillary electrophoresis CF-FAB mass spectrometric analysis of DNA adducts. Anal. Chem. 67, 891–900.CrossRefPubMedGoogle Scholar
  28. 28.
    Willems, A. V., Deforce, D. L., Van Peteghem, C. H., Van Bocxlaer, J. F. (2005) Analysis of nucleic acid constituents by on-line capillary electrophoresis-mass spectrometry. Electrophoresis 26, 1221–1253.CrossRefPubMedGoogle Scholar
  29. 29.
    Willems, A. V., Deforce, D. L., Van Peteghem, C. H., Van Bocxlaer, J. F. (2005) Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization quadrupole time-of-flight mass spectrometry. Electrophoresis 26, 1412–1423.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • An Willems
    • 1
  • Dieter L. Deforce
    • 1
  • Jan Van Bocxlaer
    • 1
  1. 1.Laboratory of Medical Biochemisty and Clinical AnalysisGhent UniversityGhentBelgium

Personalised recommendations