A Multiplexed Protein Kinase Assay

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, ph...

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Bibliographic Details
Published in:Chembiochem : a European journal of chemical biology 2007-05, Vol.8 (8), p.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: Article
Language:English
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
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Summary: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.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.200600522