Alkylation kinetics of proteins in preparation for two-dimensional maps: A matrix assisted laser desorption/ionization-mass spectrometry investigation

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Abstract
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.

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