Protein kinase peptide substrate determination using peptide...

Details Protein kinase peptide substrate determination using peptide... Protein kinase peptide substrate determination using peptide...

C - Chemistry, Metallurgy

12

N

C12N 9/12 (2006.01) C07K 7/08 (2006.01) C12Q 1/48 (2006.01)

Patent

CA 2387406

A method of isolating and identifying peptide substrates for a protein kinase is disclosed. The method involves a combination of size exclusion and gallium-based metal affinity chromatography. The method includes the steps of incubating a protein kinase with a peptide library in the presence of kinase reaction components, the library comprising library members; separating library members from the kinase reaction components using size exclusion chromatography to give a pool of phosphopeptides and unphosphorylated peptides; contacting the pool with immobilized gallium ions to form chelated phosphopeptides; eluting chelated phosphopeptides away from the gallium ions to give eluted phosphopeptides; sequencing the eluted phosphopeptides, whereby a preferred amino acid sequence of a preferred peptide substrate for a protein kinase is elucidated. Also disclosed is a method of identifying a compound that modulates the protein kinase catalyzed phosphorylation of a peptide substrate and a method of designing protein kinase substrates.

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