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

Abstract

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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Acknowledgments

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). https://doi.org/10.1007/s13361-014-0873-4

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Keywords

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