title: A multiplexed protein kinase assay.
abstract: 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.
authors.affiliation: Illumina Inc, San Diego, CA 92121-1975, USA.
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Shults
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kozlov
authors.forename: Nicholas
authors.initials: N
authors.lastname: Nelson
authors.forename: Bahram G
authors.initials: BG
authors.lastname: Kermani
authors.forename: Peter C
authors.initials: PC
authors.lastname: Melnyk
authors.forename: Veronika
authors.initials: V
authors.lastname: Shevchenko
authors.forename: Anu
authors.initials: A
authors.lastname: Srinivasan
authors.forename: Joseph
authors.initials: J
authors.lastname: Musmacker
authors.forename: John P
authors.initials: JP
authors.lastname: Hachmann
authors.forename: David L
authors.initials: DL
authors.lastname: Barker
authors.forename: Michal
authors.initials: M
authors.lastname: Lebl
authors.forename: Chanfeng
authors.initials: C
authors.lastname: Zhao
mesh_terms: 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
chemical_list: D002457:Cell Extracts; D009841:Oligonucleotides; D010455:Peptides; D012996:Solutions; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D004247:DNA; D011494:Protein Kinases