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 journalArticle

81 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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    McCarthy, J., Hopwood, F., Oxley, D., Laver, M., Castagna, A., Righetti, P. G., ... Herbert, B. (2003). Carbamylation of proteins in 2-D electrophoresis - Myth or reality? Journal of Proteome Research, 2(3), 239-242. https://doi.org/10.1021/pr025564b