Characterization of SDS-PAGE-separated proteins by matrix-assisted laser desorption ionization mass spectrometry

A new strategy to characterize SDS-PAGE-separated proteins with MALDI MS is described. The proteins, electroblotted onto nitrocellulose after SDS-PAGE separation and stained with reversible Ponceau S dye, are readily recovered by dissolving the membrane in matrix solutions prepared with acetone. The resulting mixtures are amenable to direct MALDI MS analysis, which provides a rapid and accurate means of measuring the molecular weights of SDS-PAGE-separated proteins and of peptides that result from CNBr digestion of proteins on the nitrocellulose membrane. Compared with the traditional elution method, this procedure provides more efficient detection of proteins and peptides, especially the higher molecular weight proteins from the membrane. As little as 3.5 pmol of lysozyme and 15 pmol of bovine albumin loaded onto a gel can be detected using this method. The detection sensitivity is higher than or comparable to that of the traditional Coomassie Brilliant Blue staining procedure.