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

Abstract

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 alpha- and beta-globin chains, which possess only one (alpha-) or two (beta-) -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 pI, 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.

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

“…Thiourea might have enhanced the solublization of the membranes or highly hydrophobic proteins in our samples, especially after mild sonication [22]. The solublization of membrane proteins is still the most challenging step in 2-DE-based proteomics approaches [23][24][25]. Nevertheless, the optimized protocol in this study resulted in a uniform spot distribution on a single protein map and we observed excellent reproducibility (Fig.…”

Calcyclin, a Ca2+ Ion-binding Protein, Contributes to the Anabolic Effects of Simvastatin on Bone

“…The samples were loaded onto the target plate using the three layers method (19). 0.6 l of matrix solution (10 mg/ml sinapinic acid in 60% acetonitrile, 40% water, 0.1% trifluoroacetic acid, prepared fresh every day) were loaded onto the sample plates and left to dry at room temperature.…”

Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation

“…Homogenates were left in lysis buffer for 1 h and centrifuged at 14 000 r.p.m. Supernatants were collected, reduced and alkylated prior to isoelectrofocusing as previously described, 39 lyophilized, resuspended in rehydration buffer and stored at À801C until the isoelectrofocusing procedure. To release the DNA-bound/detergent-insoluble PCNA fraction, neoplastic and cirrhotic sample pellets were digested with DNAse-I as previously reported.…”