A Multiplexed Protein Kinase Assay

• Published in: Chembiochem : a European journal of chemical biology Vol. 8; no. 8; pp. 933 - 942
• Main Authors: Shults, Melissa D., Kozlov, Igor A., Nelson, Nicholas, Kermani, Bahram G., Melnyk, Peter C., Shevchenko, Veronika, Srinivasan, Anu, Musmacker, Joseph, Hachmann, John P., Barker, David L., Lebl, Michal, Zhao, Chanfeng
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 25-05-2007; WILEY‐VCH Verlag
• Subjects: analytical methods; Cell Extracts; DNA - chemistry; Enzyme Activation; enzymes; HeLa Cells; Humans; Isotope Labeling; microarrays; Molecular Probe Techniques; Oligonucleotides - chemistry; Peptides - chemistry; Phosphorylation; Phosphoserine - chemistry; Phosphothreonine - chemistry; Phosphotyrosine - chemistry; Protein Kinases - analysis; Protein Kinases - chemistry; proteomics; Sensitivity and Specificity; Solutions - chemistry; Substrate Specificity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200600522

We report a novel protein kinase assay designed for high‐throughput detection of one or many kinases in a complex mixture. A solution‐phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.
High‐throughput detection of one or many kinases has been achieved in complex mixtures. Peptide substrates are encoded by a unique oligonucleotide tag to allow solution‐phase phosphorylation. A novel, universal phosphate‐labeling method enables readout of the phosphorylation state of each peptide on a microarray.