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

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,...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2015-12, Vol.407 (30), p.8999
Main Authors: Zigoneanu, Imola G, Sims, Christopher E, Allbritton, Nancy L
Format: Article
Language:English
Subjects:
Amino acids
Analysis
Capillary electrophoresis
Chemical properties
Cytology
Electrophoresis
Kinases
Lasers
Mass spectrometry
NMR
Nuclear magnetic resonance
Peptides
Polyethylene glycol
Protein kinases
Proteins
Proteolysis
Scientific imaging
Substrates
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Summary: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 H.sub.3PO.sub.4, 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.
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-015-9065-8