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Negative ion fragmentations of disulfide-containing cross-linking reagents are competitive with aspartic acid side-chain-induced cleavages

Authors

  • Antonio N. Calabrese,

    1. School of Chemistry and Physics, The University of Adelaide, Adelaide, SA, Australia
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    • These authors contributed equally to this work.

  • Tianfang Wang,

    1. School of Chemistry and Physics, The University of Adelaide, Adelaide, SA, Australia
    2. School of Science, Education and Engineering, The University of the Sunshine Coast, Sippy Downs, Qld, Australia
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    • These authors contributed equally to this work.

  • John H. Bowie,

    1. School of Chemistry and Physics, The University of Adelaide, Adelaide, SA, Australia
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  • Tara L. Pukala

    Corresponding author
    • School of Chemistry and Physics, The University of Adelaide, Adelaide, SA, Australia
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T. L. Pukala, School of Chemistry and Physics, The University of Adelaide, Adelaide, SA, Australia 5005.

E-mail: tara.pukala@adelaide.edu.au

Abstract

RATIONALE

It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture.

METHODS

Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages.

RESULTS

Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied, only those from aspartic acid compete significantly with those of the cross-linker disulfide.

CONCLUSIONS

Low-energy cleavage processes from aspartic acid that compete with cross-linker fragmentations occur in the negative ion MS/MS spectra of the cross-linked peptides, irrespective of the spacer arm length. Other fragmentation pathways do not significantly interfere with low-energy disulfide cleavage, making the presence of additional product ions in the MS/MS spectrum diagnostic for the presence of aspartic acid. Copyright © 2012 John Wiley & Sons, Ltd.

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