A novel polyacrylamide gel system for proteomic use offering controllable pore expansion by crosslinker cleavage.
SDS-PAGE is still one of the most widespread separation techniques in proteomic research and usually coupled to subsequent MS measurement for protein identification. The proteins are digested while embedded in the gel matrix. The resultant peptides are eluted out of the gel and finally analyzed. The in-gel digestion process suffers from several drawbacks which influence the experimental outcome with respect to protein sequence coverage and detection sensitivity. Limited accessibility of the protease to the substrate protein and insufficient peptide extraction represent the two major problems. To specifically target these issues, we established a novel partly reversible gel system, in which the gel matrix can be conditionally cleaved to increase the pore diameters. By using a crosslinker mixture consisting of Bis and ethylene-glycol-diacrylate the acrylamide filament interconnections can be partly hydrolyzed in alkaline solution. The new hybrid gels have been tested to be compatible with a variety of acidic staining techniques. They exhibit similar electrophoretic performance compared with regular solely Bis-based gels, but yield significantly better MS results. Thus, the Bis/ethylene-glycol-diacrylate SDS-PAGE gel system is a promising alternative for MS-based in-gel workflows and might be transferred to other gel-electrophoretic applications.
Bornemann
Sandra
S
Cluster of Excellence Macromolecular Complexes, Institute for Pharmaceutical Chemistry, Goethe-University, D-60438 Frankfurt am Main, Germany.
Rietschel
Benjamin
B
Baltruschat
Sabrina
S
Karas
Michael
M
Meyer
Bjoern
B
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005026:Ethylene Glycols / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D016062:Porosity; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D000516:alpha-Amylases / Q000737:chemistry
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005026:Ethylene Glycols; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D000516:alpha-Amylases