Electrophoretic Isolation /Characterization--Biological

Child Health And Human Development

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Abstract

Proteomics rests on protein identification by mass, i.e. mass spectrometry (MS). Proteins occurring in the mixture of a biological material can be identified by a global MS analysis of their proteolytic fragments if the DNA sequences in that material have been elucidated and data bases of those sequences are available (e.g. the human genome in the analysis of human proteins). If that is not the case, it is necessary to separate the protein mixture prior to MS, usually by 2-D-PAGE. The conventional procedure for that purpose consists of locating the protein of interest on the 2D-gel pattern, identifying its position by image analysis and excision of the protein spot of interest followed by its dehydration, reswelling in presence of trypsin, and elution of the tryptic peptides derived from the protein for MS analysis. An alternative procedure of collecting the protein identified as a gel band (or spot) developed in this Laboratory replaces the band excision by direct electroelution of the protein by placing electrodes across the band at a direction orthogonal to that of electrophoretic migration in the separating gel. That procedure has advantages over the conventional one: I) it allows for MS of the protein prior to that of its peptides; ii) it allows for electrofocusing (IEF) and re-electrophresis analysis of the eluate prior to MS; iii) it avoids the presence of gel during tryptic digestion thus increasing its rate and avoiding adsorption of protein, peptides and enzyme. Iv) it appears more adaptable to automation of sequential spot elution than an excision procedure. We were able to verify items I) and ii). Items iii) and iv) remain hypothetical. Sequential electroelution of protein bands remains to be solved. A possible solution appeared to rest on the polymerization of gels on a net which is permeable to the orthogonally directed current of electroelution. Although nets covalently linkable to polyacrylamide are commercially available, a cassette designed and constructed for that purpose failed to prevent leakage of the polymerization mixture through the gel. A re-design is in progress. Another problem of the net-approach is the fluorescence of Nylon which obliterates the fluorescent signal on which protein detection is based. We are experimenting with polypropylene nettings in the expectation to at least greatly reduce the fluorescence intensity of the net, since GelBond (a modified polypropylene) does not interfere with fluorescent detection of protein. Once these net problems are solved, we intend to position the gel by moving the net support under micromanipulator control; this will avoid gel stretching, as we move sequentially from spot to spot. A suitable frame for the net will be constructed. Once this is done, the positioning of the net will have to be interfaced with the image analysis system to arrive at a computer controlled automated protein elution method without any gel sectioning. The primary potential importance of that approach, compared with the conventional one, consists of the fact that the protein is obtained, not its peptides. This will allow for a fundamental change in 2-D technique, which presently only yields the 10-20% of the proteins which are the most abundant, and which in the usual practice suffers from the presence of charge isomeric forms under the SDS-PAGE band in view of the inefficient 1st dimension (excessive pI-range, insufficient volt-hours). The electroelution of the SDS-protein will allow one to replace that 1st dimension with IEF-runs within narrow pI-ranges and with any desired protein load, so that minor bands are revealed. Of further interest for the future of proteomics is the fact that we have achieved a direct electroelution of a native protein from a non-detergent gel. This finding foreshadows the possibility of identifying post-translationally modified proteins which on SDS-PAGE migrate together. CZE is the electrophoretic separation method suited for particles which are too large to enter into gels. A major problem in the CZE separations of such particles has been their adsorption onto the inner silanol walls of the capillary which are coated conventionally with polyacrylamide to reduce electroosmosis. However, the walls coated with either polyacrylamide or a chemical relative of it, EPDMA, exhibit peak spreading due to adsorption which greatly reduces the resolving power of the CZE separation of large particles. An attempt to solve that problem by making the coating agent more hydrophilic (to reduce hydrophobic adsorption) failed in application to large particles due to an antagonism between the capacity to bind to silanol and the hydrophilicity of the coating agent (presumably due to charge repulsion). The expected improvement of resolution was only observed with mildly hydrophilic coating agents in application to the CZE of proteins.