title: Separation of peptide fragments of a protein kinase C substrate fused to a β-hairpin by capillary electrophoresis.
abstract: Synthetic peptides incorporating well-folded β-hairpin peptides possess advantages in a variety of cell biology applications by virtue of increased resistance to proteolytic degradation. In this study, the WKpG β-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 β-hairpin-conjugated substrate and its PKCα-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.
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, 27599, USA.|North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Imola G
authors.initials: IG
authors.lastname: Zigoneanu
authors.affiliation: Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, 27599, USA. nlallbri@unc.edu.|North Carolina State University, Raleigh, NC, 27695, USA. nlallbri@unc.edu.|Department of Chemistry, University of North Carolina, Chapel Hill, NC, 27599, USA. nlallbri@unc.edu.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D011493:Protein Kinase C
keywords: Capillary electrophoresis; PKC; Peptide separation; Protease resistance; Reporter; β-Hairpin