Synthetic peptide substrates for casein kinase II

Abstract

Synthetic peptide substrates for CKII are useful reagents in the analysis of phosphorylation sites when used in conjunction with biochemical and genetic analysis of the protein substrates for the enzyme. A multidisciplinary approach should be applied to the characterization of the synthetic peptide products, including amino acid analysis, sequencing, and mass spectrometry. Synthetic procedures for CKII substrate peptides often result in anisole adducts and dehydrated forms. Mass spectrometry is invaluable in identifying these contaminants, and preparative HPLC can be used to separate them from the desired product. Quantitative analysis of the CKII phosphorylation of peptides can utilize phosphocellulose paper if the peptide has a basic sequence, or thin-layer chromatography, if the peptide has no basic portion. Qualitative analysis using electrophoresis and mass spectrometry help to establish the stoichiometry of phosphorylation. Sequence analysis of phosphoserine after beta elimination and derivatization is useful in quantifying adjacent phosphorylation sites. Overall, application of a variety of techniques permits detailed analysis of CKII phosphorylation sites on synthetic peptides that are model substrates.