Carbamylation of proteins in 2-D electrophoresis - Myth or reality?

John McCarthy, Femia Hopwood, David Oxley, Matthew Laver, Annalisa Castagna, Pier Giorgio Righetti, Keith Williams, Ben Herbert*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    84 Citations (Scopus)

    Abstract

    Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.

    Original languageEnglish
    Pages (from-to)239-242
    Number of pages4
    JournalJournal of Proteome Research
    Volume2
    Issue number3
    DOIs
    Publication statusPublished - May 2003

    Keywords

    • Alkylation
    • Artifacts
    • Carbamylation
    • MALDITOF mass spectrometry
    • Proteomics
    • Reduction
    • Two-dimensional electrophoresis

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