Peptide fractionation by SDS-free polyacrylamide gel electrophoresis for proteomic analysis via DF-PAGE

Here we present a procedure for peptide fractionation by SDS-free polyacrylamide gel electrophoresis, based on discontinuous buffer systems. In the absence of SDS, peptide migration depends both on their molecular mass and on their net charge at the electrophoresis pH. By selecting the separation pH, peptide mobility is modulated. In the original discontinuous buffer system (Tris/glycine), peptides that migrate to the anode have pI values below 6.8 and distribute along the lane in a pI decreasing order, while at acidic pH, as that afforded by histidine/MOPS buffer system, peptides with pI below 5.5 are fractionated. Separation at acid pH is particularly useful for recovering phosphopeptides as well as other highly negatively charged peptides, as those containing sialic or sulfate substituents. Both separation conditions in Tris/glycine and in histidine/MOPS are applicable to proteomic studies, by dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). First, complex protein samples are separated via SDS-PAGE, and after in-gel proteolysis, peptides are loaded on a second SDS-free gel, where they are separated as described here.