Separation of peptide fragments of a protein kinase C substrate fused to a beta-hairpin by capillary electrophoresis

摘要

Synthetic peptides incorporating well-folded beta-hairpin peptides possess advantages in a variety of cell biology applications by virtue of increased resistance to proteolytic degradation. In this study, the WKpG beta-hairpin peptide fused to a protein kinase C (PKC) substrate was synthesized, and capillary-electrophoretic separation conditions for this peptide and its proteolytic fragments were developed. Fragments of WKpG-PKC were generated by enzymatic treatment with trypsin and Pronase E to produce standards for identification of degradation fragments in a cellular lysate. A simple buffer system of 250 mM H3PO4, pH 1.5 enabled separation of WKpG-PKC and its fragments by capillary electrophoresis in less than 16 min. Using a cellular lysate produced from Ba/F3 cells, the beta-hairpin-conjugated substrate and its PKC alpha-phosphorylated product could be detected and separated from peptidase-generated fragments produced in a cell lysate. The method has potential application for identification and quantification of WKpG-PKC and its fragments in complex biological systems when the peptide is used as a reporter to assay PKC activity.