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More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides


Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO ion by MS3 revealed a so far unreported consecutive excision of a metaphosphate (PO3 )-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO3 loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs.

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  1. 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. 12, 180–192 (2001)

    CAS  Article  Google Scholar 

  2. McLuckey, S.A., Habibi-Goudarzi, S.: Decompositions of multiply-charged oligonucleotide anions. J. Am. Chem. Soc. 115, 12085–12095 (1993)

    CAS  Article  Google Scholar 

  3. 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. 12, 193–205 (2001)

    CAS  Article  Google Scholar 

  4. 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. 9, 683–691 (1998)

    CAS  Article  Google Scholar 

  5. Schürch, S., Bernal-Mendez, E., Leumann, C.J.: Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides. J. Am. Soc. Mass Spectrom. 13, 936–945 (2002)

    Article  Google Scholar 

  6. Tromp, J.M., Schürch, S.: Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 16, 1262–1268 (2005)

    CAS  Article  Google Scholar 

  7. Andersen, T.E., Kirpekar, F., Haselmann, K.F.: RNA fragmentation in MALDI mass spectrometry studied by H/D-exchange: mechanisms of general applicability to nucleic acids. J. Am. Soc. Mass Spectrom. 17, 1353–1368 (2006)

    CAS  Article  Google Scholar 

  8. Tromp, J.M., Schürch, S.: Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides. Rapid Commun. Mass Spectrom. 20, 2348–2354 (2006)

    CAS  Article  Google Scholar 

  9. Keough, T., Baker, T.R., Dobson, R.L.M., Lacey, M.P., Riley, T.A., Hasselfield, J.A., Hesselberth, P.E.: Antisense DNA oligonucleotides. 2. The use of matrix-assisted laser desorption ionization mass-spectrometry for the sequence verification of methylphosphonate oligodeoxyribonucleotides. Rapid Commun. Mass Spectrom. 7, 195–200 (1993)

    CAS  Article  Google Scholar 

  10. 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. 31, 1277–1283 (1996)

    CAS  Article  Google Scholar 

  11. Wang, B.H., Hopkins, C.E., Belenky, A.B., Cohen, A.S.: Sequencing of modified oligonucleotides using in-source fragmentation and delayed pulsed ion extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Int. J. Mass Spectrom. 169, 331–350 (1997)

    Article  Google Scholar 

  12. Monn, S.T.M., Schürch, S.: New aspects of the fragmentation mechanisms of unmodified and methylphosphonate-modified oligonucleotides. J. Am. Soc. Mass Spectrom. 18, 984–990 (2007)

    CAS  Article  Google Scholar 

  13. Nyakas, A., Stucki, S.R., Schürch, S.: Tandem mass spectrometry of modified and platinated oligoribonucleotides. J. Am. Soc. Mass Spectrom. 22, 875–887 (2011)

    CAS  Article  Google Scholar 

  14. 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. 12, 580–589 (2001)

    CAS  Article  Google Scholar 

  15. Nyakas, A., Eymann, M., Schürch, S.: The influence of cisplatin on the gas-phase dissociation of oligonucleotides studied by electrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 20, 792–804 (2009)

    CAS  Article  Google Scholar 

  16. Rozenski, J.: Mongo oligonucleotide mass calculator. Available at: Accessed January 2014

  17. Rozenski, J., McCloskey, J.A.: SOS: a simple interactive program for ab initio oligonucleotide sequencing by mass spectrometry. J. Am. Soc. Mass Spectrom. 13, 200–203 (2002)

    CAS  Article  Google Scholar 

  18. Nyakas, A., Blum, L.C., Stucki, S.R., Reymond, J.-L., Schürch, S.: OMA and OPA software-supported mass spectra analysis of native and modified nucleic acids. J. Am. Soc. Mass Spectrom. 24, 249–256 (2013)

  19. Yang, J., Leopold, P., Helmy, R., Parish, C., Arvary, B., Mao, B., Meng, F.: Design and application of an easy to use oligonucleotide mass calculation program. J. Am. Soc. Mass Spectrom. 24, 1315–1318 (2013)

    CAS  Article  Google Scholar 

  20. Pan, S., Verhoeven, K., Lee, J.K.: Investigation of the initial fragmentation of oligodeoxynucleotides in a quadrupole ion trap: charge level-related base loss. J. Am. Soc. Mass Spectrom. 16, 1853–1865 (2005)

    CAS  Article  Google Scholar 

  21. Huang, T.Y., Kharlamova, A., Liu, J., McLuckey, S.A.: Ion trap collision-induced dissociation of multiply deprotonated RNA: c/y-ions versus (a-B)/w-ions. J. Am. Soc. Mass Spectrom. 19, 1832–1840 (2008)

    CAS  Article  Google Scholar 

  22. Rice, J.M., Dudek, G.O., Barber, M.: Mass spectra of nucleic acid derivatives—pyrimidines. J. Am. Chem. Soc. 87, 4569–4576 (1965)

    CAS  Article  Google Scholar 

  23. Jochims, H.W., Schwell, M., Baumgartel, H., Leach, S.: Photoion mass spectrometry of adenine, thymine and uracil in the 6-22 eV photon energy range. Chem. Phys. 314, 263–282 (2005)

    CAS  Article  Google Scholar 

  24. Flosadóttir, H.D., Jónsson, H., Sigurdsson, S.T., Ingólfsson, O.: Experimental and theoretical study of the metastable decay of negatively charged nucleosides in the gas phase. Phys. Chem. Chem. Phys. 13, 15283–15290 (2011)

    Article  Google Scholar 

  25. Almeida, D., Antunes, R., Martins, G., Eden, S., da Silva, F.F., Nunes, Y., Garcia, G., Limao-Vieira, P.: Electron transfer-induced fragmentation of thymine and uracil in atom-molecule collisions. Phys. Chem. Chem. Phys. 13, 15657–15665 (2011)

    CAS  Article  Google Scholar 

  26. Imhoff, M., Deng, Z.W., Huels, M.A.: Identification of ion fragments produced from thymine and deuterated thymine by low energy ion impact in films and electron impact in the gas phase. Int. J. Mass Spectrom. 245, 68–77 (2005)

    CAS  Article  Google Scholar 

  27. Nelson, C.C., McCloskey, J.A.: Collision-induced dissociation of uracil and its derivatives. J. Am. Soc. Mass Spectrom. 5, 339–349 (1994)

    CAS  Article  Google Scholar 

  28. Cao, H., Wang, Y.: Collisionally activated dissociation of protonated 2'-deoxycytidine, 2'-deoxyuridine, and their oxidatively damaged derivatives. J. Am. Soc. Mass Spectrom. 17, 1335–1341 (2006)

    CAS  Article  Google Scholar 

  29. Kamel, A.M., Munson, B.: Collisionally-induced dissociation of substituted pyrimidine antiviral agents: mechanisms of ion formation using gas phase hydrogen/deuterium exchange and electrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 18, 1477–1492 (2007)

    CAS  Article  Google Scholar 

  30. Bald, I., Flosadóttir, H.D., Ómarsson, B., Ingólfsson, O.: Metastable fragmentation of a thymidine-nucleotide and its components. Int. J. Mass Spectrom. 313, 15–20 (2012)

    CAS  Article  Google Scholar 

  31. Arani, L.S., Mignon, P., Abdoul-Carime, H., Farizon, B., Farizon, M., Chermette, H.: DFT study of the fragmentation mechanism of uracil RNA base. Phys. Chem. Chem. Phys. 14, 9855–9870 (2012)

    CAS  Article  Google Scholar 

  32. Guillaumont, S., Tortajada, J., Salpin, J.Y., Lamsabhi, A.M.: Experimental and computational study of the gas-phase interactions between lead(II) ions and two pyrimidic nucleobases: uracil and thymine. Int. J. Mass Spectrom. 243, 279–293 (2005)

    CAS  Article  Google Scholar 

  33. Flosadóttir, H.D., Ómarsson, B., Bald, I., Ingólfsson, O.: Metastable decay of DNA components and their compositions—a perspective on the role of reactive electron scattering in radiation damage. Eur. Phys. J. D 66, 13 (2012)

    Article  Google Scholar 

  34. Lamsabhi, A.M., Alcami, M., Mo, O., Yanez, M., Tortajada, J., Salpin, J.-Y.: Unimolecular reactivity of uracil-Cu2(+) complexes in the gas phase. Chem. Phys. Chem. 8, 181–187 (2007)

    CAS  Article  Google Scholar 

  35. Beach, D.G., Gabryelski, W.: Revisiting the reactivity of uracil during collision induced dissociation: tautomerism and charge-directed processes. J. Am. Soc. Mass Spectrom. 23, 858–868 (2012)

    CAS  Article  Google Scholar 

  36. Improta, R., Scalmani, G., Barone, V.: Radical cations of DNA bases: some insights on structure and fragmentation patterns by density functional methods. Int. J. Mass Spectrom. 201, 321–336 (2000)

    CAS  Article  Google Scholar 

  37. da Silva, F.F., Matias, C., Almeida, D., Garcia, G., Ingólfsson, O., Flosadóttir, H.D., Ómarsson, B., Ptasinska, S., Puschnigg, B., Scheier, P., Limao-Vieira, P., Denifl, S.: NCO, a key fragment upon dissociative electron attachment and electron transfer to pyrimidine bases: site selectivity for a slow decay process. J. Am. Soc. Mass Spectrom. 24, 1787–1797 (2013)

    Article  Google Scholar 

  38. Ziehe, M., Grossmann, T.N., Seitz, O., Linscheid, M.W.: New aspects in fragmentation of peptide nucleic acids: comparison of positive and negative ions by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun. Mass Spectrom. 23, 1132–1138 (2009)

    CAS  Article  Google Scholar 

  39. Bohringer, M., Roth, H.J., Hunziker, J., Gobel, M., Krishnan, R., Giger, A., Schweizer, B., Schreiber, J., Leumann, C., Eschenmoser, A.: Why pentose and not hexose nucleic-acids. 2. Preparation of oligonucleotides containing 2',3'-dideoxy-beta-d-glucopyranosyl building-blocks. Helv. Chim. Acta 75, 1416–1477 (1992)

    Article  Google Scholar 

  40. Anusiewicz, W., Berdys-Kochanska, J., Czaplewski, C., Sobczyk, M., Daranowski, E.M., Skurski, P., Simons, J.: Charge loss in gas-phase multiply negatively charged oligonucleotides. J. Phys. Chem. A 109, 240–249 (2005)

    CAS  Article  Google Scholar 

  41. Chandra, A.K., Nguyen, M.T., Uchimaru, T., Zeegers-Huyskens, T.: Protonation and deprotonation enthalpies of guanine and adenine and implications for the structure and energy of their complexes with water: comparison with uracil, thymine, and cytosine. J. Phys. Chem. A 103, 8853–8860 (1999)

    CAS  Article  Google Scholar 

  42. Stucki, S.R., Desiron, C., Nyakas, A., Marti, S., Leumann, C.J., Schürch, S.: Gas-phase dissociation of homo-DNA oligonucleotides. J. Am. Soc. Mass Spectrom. 24, 1997–2006 (2013)

    CAS  Article  Google Scholar 

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The authors thank C. Désiron and C. J. Leumann for providing the homoDNA sequences. Further, the authors gratefully acknowledge financial support of this work by the Swiss National Science Foundation (grant #200020_140628).

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Correspondence to Stefan Schürch.

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Adrien Nyakas and Rahel P. Eberle contributed equally to this project.

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Nyakas, A., Eberle, R.P., Stucki, S.R. et al. More Than Charged Base Loss — Revisiting the Fragmentation of Highly Charged Oligonucleotides. J. Am. Soc. Mass Spectrom. 25, 1155–1166 (2014).

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  • Oligonucleotides
  • Tandem mass spectrometry
  • Fragmentation
  • CID
  • Nucleic acids
  • Dissociation mechanism
  • Modified oligonucleotides