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

The standard procedure adopted up to the present in proteome analysis calls for just reduction prior to the isoelectric focusing/immobilized pH gradie nt (IEF/IPG) step, followed by a second reduction/alkylation step in betwee n the first and second dimension, in preparation for the sodium dodecyl sul fate-polyacrylamide gel electrophoresis (SDS-PAGE) step. This protocol is f ar from being optimal. It is here demonstrated, by matrix assisted laser de sorption/ionization-time of flight (MALDI-TOF)-mass spectrometry, that fail ure to reduce and alkylate proteins prior to any electrophoretic step (incl uding the first dimension) results in a large number of spurious spots in t he 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 simpl e polypeptides such as the human alpha- and P-globin chains, which possess only one (alpha-) or two (beta-) -SH groups. As a result, misplaced spots a re to be found in the resulting two-dimensional (2-D) map, if performed wit h the wrong protocol. The number of such artefactual spots can be impressiv ely 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 s ubstantial loss of spots in the alkaline gel region, possibly due to the fa ct that these proteins, at their pi, regenerate their disulfide bridges wit h 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.