Alkylation kinetics of proteins in preparation for two-dimensional maps

A matrix assisted laser desorption/ionization-mass spectrometry investigation

Marina Galvani, Mahmoud Hamdan, Ben Herbert, Pier Giorgio Righetti*

*Corresponding author for this work

Research output: Contribution to journalArticle

83 Citations (Scopus)


All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached ∼ 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

Original languageEnglish
Pages (from-to)2058-2065
Number of pages8
Issue number10
Publication statusPublished - 2001


  • Alkylation
  • Ionization-time of flight-mass spectrometry
  • Matrix assisted laser desorption
  • Reduction
  • Two-dimensional maps

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