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, 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.
Shults
Melissa D
MD
Illumina Inc, San Diego, CA 92121-1975, USA.
Kozlov
Igor A
IA
Nelson
Nicholas
N
Kermani
Bahram G
BG
Melnyk
Peter C
PC
Shevchenko
Veronika
V
Srinivasan
Anu
A
Musmacker
Joseph
J
Hachmann
John P
JP
Barker
David L
DL
Lebl
Michal
M
Zhao
Chanfeng
C
D002457:Cell Extracts; D004247:DNA / Q000737:chemistry; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D015336:Molecular Probe Techniques; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D019000:Phosphotyrosine / Q000737:chemistry; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D013379:Substrate Specificity
D002457:Cell Extracts; D009841:Oligonucleotides; D010455:Peptides; D012996:Solutions; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D004247:DNA; D011494:Protein Kinases