Prefractionation techniques in proteome analysis: The mining tools of the third millennium

Abstract

The present review deals with prefractionation protocols used in proteomic investigation in preparation for mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) map analysis. Briefly, reported methods focus on cell organelle differential centrifugation and on chromatographic approaches, to continue in extenso with a panoply of electrophoretic methods. In the case of chromatography, procedures useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins, revealed several hundreds of new species, previously undetected in unfractionated samples. Novel chromatographic prefractionation methods are also discussed such as a multistaged fractionation column, consisting in a set of immobilized chemistries, serially connected in a stack format (an assembly of seven blocks), each capable of harvesting a given protein population. Such a method significantly simplifies the complexity of treated samples while concentrating species, all resulting in a larger number of visible proteins by MS or 2-DE. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separation are multichamber apparatus, such as the multicompartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads. Multicup device electrophoresis and several others, exploiting the conventional technique of carrier ampholyte focusing, are reviewed. This review also reports approaches for sample treatments in order to detect low-abundance species. Among others, a special emphasis is made on the reduction of concentration difference between proteins constituting a sample. This latter consists in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of 20 amino acids, resulting in millions of different structures. When these beads are impregnated with complex proteomes (e.g., human sera) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility to evidence low-abundance species. 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".

Enabling Coupled Quantitative Genomics and Proteomics Analyses from Rat Spinal Cord Samples

“…Membrane Versus Soluble Protein Abundance-Sample prefractionation has been extensively demonstrated to improve the resolution and detection of proteins (24). Here well characterized methods of separating the homogenized tissue into total membrane and soluble proteomes were tested for their applicability in the analysis of spinal cord segments (18).…”

“…size, density, and cellular localization), chemically (e.g. hydrophobicity, charge properties, and specific proteins), or otherwise defined fractions prior to resolving proteins in each of these separate fractions (24). Chief among the advantages of this approach is improved resolution and detection of low abundance proteins: many proteins that fall below detection sensitivity in separations of the total proteome are effectively concentrated by virtue of the sample prefractionation strategy and are thus detectable in an optimized separation of the defined fraction.…”

Vascular proteomics

“…[104]. The challenge is thus to reach the remaining proteome comprising only 1% of the plasma proteome [105]. Further complicating the study of the human plasma proteome are temporal and spatial dynamics.…”

Proteomics‐driven progress in neurodegeneration research

“…In combination with the visualization methods, sophisticated software is used for protein identification and quantification [14][15][16][17][18][19][20]. Separation is usually performed by 2-DE or multidimensional LC often following prefractionation of the original protein sample [21][22][23][24]. 2-DE comprises two steps (dimensions): separation of the proteins on the basis of differences in their net charge by IEF and separation of the focused proteins on the basis of differences in their molecular masses by SDS polyacrylamide gels [25][26][27][28].…”