Reduction of alkylation of proteins in preparation of two-dimensional map analysis: Why, when, and how?

Ben Herbert, Marina Galvani, Mahmoud Hamdan, Erna Olivieri, John MacCarthy, Sanne Pedersen, Pier Giorgio Righetti*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    57 Citations (Scopus)


    The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradient (IEF/IPG) step, followed by a second reduction/alkylation step in between the first and second dimension, in preparation for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is far from being optimal. It is here demonstrated, by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that failure to reduce and alkylate proteins prior to any electrophoretic step (including the first dimension) results in a large number of spurious spots in the alkaline pH region, due to "scrambled" disulfide bridges among like and unlike chains. This series of artefactual spots comprises not only dimers, but an impressive series of oligomers (up to nonamers) in the case of simple polypeptides such as the human α- and β-globin chains, which possess only one (α-) or two (β-) -SH groups. As a result, misplaced spots are to be found in the resulting two-dimensional (2-D) map, if performed with the wrong protocol. The number of such artefactual spots can be impressively large. In the case of analysis of complex samples, such as human plasma, it is additionally shown that failure to alkylate proteins results in a substantial loss of spots in the alkaline gel region, possibly due to the fact that these proteins, at their p/, regenerate their disulfide bridges with concomitant formation of macroaggregates which become entangled with and trapped within the polyacrylamide gel fibers. This strongly quenches their transfer in the subsequent SDS-PAGE step.

    Original languageEnglish
    Pages (from-to)2046-2057
    Number of pages12
    Issue number10
    Publication statusPublished - 2001


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


    Dive into the research topics of 'Reduction of alkylation of proteins in preparation of two-dimensional map analysis: Why, when, and how?'. Together they form a unique fingerprint.

    Cite this