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".

Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines

“…The difficulty of analyzing the proteome is that many low-abundance proteins are not detected due to a predicted dynamic range of up to five orders of magnitude [28]. Therefore, the field of proteomics requires the development of effective fractionation and separation methods that offer high sensitivity and wide dynamic range to detect more proteins in a cell or tissue [29]. Liquid isoelectric focusing (LIEF) as the prefractionation technology was used to crudely separate sample prior to 2-D-LC-MS/MS [30].…”

Proteomic technology for biomarker profiling in cancer: an update

“…For example, 2D gel electrophoresis suffers from low sensitivity, low dynamic range (10 3 ), difficulty in resolving proteins with extreme masses or isoelectric points, and the inability to resolve proteins with low solubility (Ong and Mann, 2005;Hanash, 2001). Some improvements were obtained when 2D methods are combined with prefractionation chromatography techniques (Righetti et al, 2003). The introduction of differential gel electrophoresis (DIGE) has provided improvement over standard 2D.…”

Teaching expression proteomics: From the wet‐lab to the laptop

“…As it is virtually impossible to separate with high resolution the whole proteome in a single 2-DE, prefractionation methods such as those applied to the isolation of organelles [11], to hydrophobic protein extraction [12,13], to sequential extraction of proteins using different extraction buffers containing progressively stronger solubilizing agents [14], and chromatographic procedures for sample enrichment [15] are described. These approaches and their advantages and limitations in quantitative expression proteomics are discussed with the students.…”