Amphoteric, buffering chromatographic beads for proteome prefractionation. I: Theoretical model

The possibility is reported here of fractionating proteins on amphoteric, buffering resins via ion‐exchange chromatography. A given protein's adsorption to a particular amphoteric buffering resin is characterized by a bell‐shaped curve in which the maximum protein binding capacity is observed at an optimum pH value lying approximately midway between the isoelectric point values (p I ) of the resin and the protein. On either side of this maximum the protein binding capacity declines steadily, reaching zero at the p I of either the protein or exchanger. For instance, on beads of p I equal to 8, four proteins, two acidic (bovine albumin and ovalbumin) and two basic (cytochrome  c and lysozyme), exhibit binding curves reaching zero values for the whole set when the exchanger is conditioned at pH 8.0. Away from the p I , and on both sides of the pH scale, the bell‐shaped adsorption curves reach a maximum, for each protein, at a pH located at the midpoint between the p I values of each protein and that of the exchanger, and decline steadily to reach zero at the p I value of each protein species. Separation of model proteins using different amphoteric buffering resins of various p I was possible at different pH values according to both the p I of the proteins and of the exchangers. It was also demonstrated, using surface enhanced laser desorption/ionization mass spectrometry and two dimensional electrophoretic mapping, that separation of an Escherichia coli cell lysate on columns packed with amphoteric buffering resins of different p I and titrated to a particular pH value, delivered two distinctly different fractions, i.e. characteristically composed of, on the one hand, proteins having a p I below the buffer pH (the ‘adsorbed’ fraction), and on the other, of alkaline proteins possessing a p I above the pH of the buffer (the ‘unadsorbed’ fraction). This approach represents an attractive addition and/or alternative to the armory of protein pre‐fractionation techniques currently employed in proteomics.