Double Fractionation by Polyacrylamide Gel Electrophoresis (DF-PAGE): new method for quantitative proteomics studies.

In order to increase the possibilities of detecting low abundance proteins in complex mixtures by proteomics techniques, it is essential to use protein or peptide separation methods to reduce the complexity of the biological sample. However, despite the wide use of several electrophoretic techniques, the usefulness of zone electrophoresis for the fractionation of complex peptide mixtures had not been previously investigated. In this work, a new method was established for proteomics studies, called Double Fractionation by PAGE (DF-PAGE). It combines protein fractionation by SDS-PAGE, in-gel enzymatic hydrolysis and peptide separation by SDS-free PAGE; the latter applied for the first time for fractionation and simplification of complex peptide mixtures. Then, it was necessary to design, for the case of DF-PAGE, a new batch buffer system for the selection of acid peptides (pI ≤ 5.5). DF-PAGE allowed the identification of a greater number of proteins than PAGE-SDS and isoelectric focusing in solution. Its application to the characterization of the active principle of the VA-MENGOC-BC® vaccine allowed the identification of 67 proteins previously undetected with traditional techniques. A series of proteins differentially modulated by the antitumor peptide CIGB-552 in the HT-29 cell line of colon adenocarcinoma were also identified by DF-PAGE, which contributed to the characterization of the molecular bases of the action of said peptide. This work granted the Annual Award of the National Academy of Sciences of Cuba for the year 2015. [ABSTRACT FROM AUTHOR]