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Desulfurization of cysteine-containing peptides resulting from sample preparation for protein characterization by mass spectrometry

Authors

  • Zhouxi Wang,

    1. Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
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  • Tomas Rejtar,

    1. Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
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  • Zhaohui Sunny Zhou,

    1. Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
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  • Barry L. Karger

    Corresponding author
    1. Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
    • Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
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Abstract

In this study, we have examined two cysteine modifications resulting from sample preparation for protein characterization by mass spectrometry (MS): (1) a previously observed conversion of cysteine into dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine into alanine. Using model peptides, the conversion of cysteine into dehydroalanine via β-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37°C, pH 7.0–9.0) without disulfide reduction and alkylation. Furthermore, the surprising conversion of cysteine into alanine was shown to occur by heating cysteine-containing peptides in the presence of a phosphine (tris(2-carboxyethyl)phosphine hydrochloride (TCEP)). The formation of alanine from cysteine, investigated by performing experiments in H2O or D2O, suggested a radical-based desulfurization mechanism unrelated to β-elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. Copyright © 2010 John Wiley & Sons, Ltd.

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