A pI-based protein fractionation method using solid-state buffers.

The analysis of very complex proteomes is dependent on efficient fractionation methods with low level of carry over from fraction to fraction. Among various possibilities the separation by ranges of isoelectric points for further analysis appears as attractive, but current methods involving an electrically driven migration in the presence of ampholyte carriers are not exempt of technical complications. In the present work a new separation concept is described involving the use of so-called solid-state buffers, in association with ion exchangers, to separate protein categories of different pI ranges with a low level of protein overlapping. Resin blends packed in separated columns are used under a cascade configuration of increasing or decreasing pH and, once proteins of different pI are adsorbed by individual resin blends, the columns are dissociated. From each column protein mixtures corresponding to a given pI range are collected by competitive desorption with salts so as to be ready for proteomic analysis. The process is rapid and does not involve electrical fields nor addition of carrier ampholyte material. The presence of potassium chloride during the separation prevents protein precipitation at the vicinity of their isoelectric points. The fractions thus obtained can be used for two dimensional electrophoresis and mass spectrometry analysis after the removal of salts.

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 (pI) of the resin and the protein. On either side of this maximum the protein binding capacity declines steadily, reaching zero at the pI of either the protein or exchanger. For instance, on beads of pI 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 pI, 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 pI values of each protein and that of the exchanger, and decline steadily to reach zero at the pI value of each protein species. Separation of model proteins using different amphoteric buffering resins of various pI was possible at different pH values according to both the pI 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 pI and titrated to a particular pH value, delivered two distinctly different fractions, i.e. characteristically composed of, on the one hand, proteins having a pI below the buffer pH (the 'adsorbed' fraction), and on the other, of alkaline proteins possessing a pI 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.

Isoelectric beads for proteome pre-fractionation. II: experimental evaluation in a multicompartment electrolyzer.

Proteome pre-fractionation in multicompartment electrolyzers is proposed here, with substantial modifications as compared to the standard technique. First of all, the classical isoelectric, buffering membranes, delimiting each compartment and acting, in pairs, as isoelectric traps, have been replaced by isoelectric buffering beads, operating on the same principle, but allowing unhindered migration of proteins (lack of sieving properties, contrary to typical continuous membrane barriers). Secondly, the isoelectric beads are not made in the conventional manner, with ionic acrylamide derivative monomers throughout their central core, but are composed of a hard, ceramic core, coated with an amphoteric buffering polymer. This minimizes mass transfer resistance of proteins that are transiently adsorbed onto the beads. As a result, significantly reduced separation times, of the order of ca. 3 h, are required for developing steady-state patterns, as compared to the lengthy times (overnight and much longer) in conventional multicompartment electrolyzers operating with isoelectric membranes. Examples of separation of standard marker proteins, as well as entire Escherichia coli lysates and human serum proteins, are given. The obtained fractions are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by surface enhanced laser desorption/ionization mass spectrometry.

2-Mercapto-5-benzimidazolesulfonic acid: an effective multimodal ligand for the separation of antibodies.

The report describes the use of 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as a ligand for the separation of antibodies by chromatography. The ligand shows a relatively specific adsorption property for antibodies from very crude biologicals at pH 5.0-5.5. At this pH range most of other proteins do not interact with the resin especially when the ionic strength is similar to physiological conditions. Several characterization studies are described such as antibody adsorption in different conditions of ionic strength, pH and temperature. These properties are advantageously used to selectively capture antibodies from very crude feed stocks without dilution or addition of lyotropic salts. Demonstration was made that the adsorption mechanism is neither based on ion exchange nor on hydrophobic associations, but rather as an assembly of a variety of properties of the ligand itself. Binding capacity in the described conditions ranges between 25 and 30 mg/mL of resin. The sorbent does not co-adsorb albumin (Alb) and seems compatible with a large variety of feedstocks. Quantitative antibody desorption occurs when the pH is raised above 8.5. The final purity of the antibody depends on the nature of the feedstock, and can reach levels of purity as high as 98%. Even with very crude biological liquids such as ascites fluids, cell culture supernatants and Chon fraction II + III from human plasma fractionation where the number of protein impurities is particularly large, immunoglobumins G (IgG) were separated at high purity level in a single step.