Prefractionation techniques in proteome analysis

Abstract

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".