title: Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities.
abstract: An assay technology for high-throughput screening of kinase and phosphatase activities is introduced. The format is based upon superquenching of fluorescent-conjugated polymers by dye-labeled kinase/phosphatase peptide substrates. The sensor platform is composed of highly fluorescent-conjugated polyelectrolytes colocated with the phosphate coordinating metal ion gallium on microspheres. Phosphorylated peptide substrates containing a quencher bind specifically to the metal ions by means of phosphate groups, resulting in quench of polymer fluorescence. The modulation of fluorescence signal is proportional to kinase or phosphatase activity and is monitored as a turn-off or turn-on signal, respectively. The assay is homogeneous and simple and can be run either as an endpoint measurement or in a kinetic mode. The assay meets the sensitivity required for high-throughput screening of kinase or phosphatase inhibitors and is a valuable tool for drug discovery. A modified version of the assay allows for the detection of protein phosphorylation.
authors.affiliation: QTL Biosystems, 2778 Agua Fria Street, Santa Fe, NM 87507, USA.
authors.forename: Frauke
authors.initials: F
authors.lastname: Rininsland
authors.forename: Wensheng
authors.initials: W
authors.lastname: Xia
authors.forename: Shannon
authors.initials: S
authors.lastname: Wittenburg
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Shi
authors.forename: Casey
authors.initials: C
authors.lastname: Stankewicz
authors.forename: Komandoor
authors.initials: K
authors.lastname: Achyuthan
authors.forename: Duncan
authors.initials: D
authors.lastname: McBranch
authors.forename: David
authors.initials: D
authors.lastname: Whitten
mesh_terms: D000595:Amino Acid Sequence; D002412:Cations; D007700:Kinetics; D008670:Metals / Q000737:chemistry*; D008863:Microspheres; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
chemical_list: D002412:Cations; D008670:Metals; D011108:Polymers; D010770:Phosphotransferases; D010744:Phosphoric Monoester Hydrolases