Two-dimensional electrophoretic characterization of microheterogeneous myelin basic protein fragments.

A new two-dimensional polyacrylamide gel electrophoresis (PAGE) system with minislab gel apparatus was devised for the rapid (4 h) analysis of peptide fragments derived from the enzymic digestion of myelin basic protein (MBP). The first dimension consisted of 5% polyacrylamide running gels in 1.9 M potassium glycinate, pH 7.3, with 4.3% stacking gels in 0.08 M potassium glycinate, pH 10.3. Anodic and cathodic buffer chambers contained 38 mM glycine/5 mM Tris, pH 8.3, and 10 mM Tris-HCl, pH 8.1, respectively. This system fractionated MBP peptides on the basis of charge. By contrast, acid-urea 15% PAGE separated MBP peptides by both charge and size. A two-dimensional system of 5% PAGE followed by sodium dodecylsulfate 15% PAGE (Laemmli) was used to resolve MBP fragments from pepsin and cathepsin D digests; this analysis indicated that cathodic mobilities could be predicted by the ratio of basic to acidic amino acids in each peptide. This method should be particularly powerful in combination with immunoblotting to identify microheterogenous fragments arising from normal and pathological metabolism of MBP.