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A revisit of high collision energy effects on collision-induced dissociation spectra using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF): application to the sequencing of RNA/DNA chimeras

Authors

  • Florence Mauger,

    1. CEA/Institut de Génomique/Centre National de Génotypage, Evry Cedex, France
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  • Jean-Claude Tabet,

    1. UPMC/IPCM (Chimie Biologique Organique et Structurale), UMR 7201, Paris Cedex 05, France
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  • Ivo G. Gut

    Corresponding author
    1. CEA/Institut de Génomique/Centre National de Génotypage, Evry Cedex, France
    2. Centro Nacional de Análisis Genómico, Barcelona, Spain
    • Correspondence to: I. G. Gut, CEA/Institut de Génomique/Centre National de Génotypage, Bâtiment G2, 2 rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France.

      E-mail: igut@pcb.ub.es

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Abstract

RATIONALE

High-energy collision-induced dissociation (CID) spectra of isomeric RNA/DNA chimeras using matrix-assisted laser desorption/ionization time-of-flight LIFT mass spectrometry (MALDI-LIFT-TOF/TOF) can potentially be applied for an exhaustive fragment characterization in a nucleic acid sequencing scheme. These chimeras contain deoxynucleotides and at the 3'-end a ribonucleotide with a 3'-phosphate group.

METHODS

Deprotonated RNA/DNA chimeras of 4-, 5-, 7- and 10-mers are analyzed by CID. This enhances consecutive dissociations from both the precursor and prompt product anions generated by MALDI and metastable fragmentations prior to entering the LIFT cell.

RESULTS

Gas-phase fragmentations of 4- and 5-mers produced many fragment ions, from base release prior to consecutive cleavage of the nucleotide phosphate bond linkage phosphate. The unusual a4 product ion is a specific and diagnostic dissociation of the 4-mer if the ribonucleotide contains cytosine. As the size of RNA/DNA chimeras increase, several abundant product ions are generated mainly from zwitterionic forms (deprotonated phosphate ester and protonated base sites): [(M-H)–BiH], [ai–BiH], wj, [wj, (ai-BiH)] (if Bi ≠ T) as internal product ion, and more rarely [wj–BiH]. The absence of the majority of the [ai–BiH] series although the wj series suggested that the higher critical energy processes with a loose transition state are favored yielding the wj series. A large number of abundant fragment ions are detected which enable each isomer to be sequenced.

CONCLUSIONS

This sequencing method is high-throughput, accurate and could be used to sequence isomers of up to 10-mers and also oligonucleotides of unknown sequence. However, RNA/DNA chimeras without thymine must be sufficiently concentrated to reach desorption of deprotonated molecular species to be selected in LIFT to produce all fragment ions within measurable abundances. Copyright © 2014 John Wiley & Sons, Ltd.

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