title: Homogeneous single-label tyrosine kinase activity assay for high throughput screening.
abstract: Protein post-translational modifications (PTMs) are regulatory mechanisms carried out by different enzymes in a cell. Kinase catalyzed phosphorylation is one of the most important PTM affecting the protein activity and function. We have developed a single-label quenching resonance energy transfer (QRET) assay to monitor tyrosine phosphorylation in a homogeneous high throughput compatible format. Epidermal growth factor receptor (EGFR) induced phosphorylation was monitored using Eu(3+)-chelate labeled peptide and label-free phosphotyrosine specific antibody in presence of a soluble quencher molecule. In the QRET kinase assay, antibody binding to phosphorylated Eu(3+)-peptide protects the Eu(3+)-chelate from luminescence quenching, monitoring high time-resolved luminescence (TRL) signals. In the presence of specific kinase inhibitor, antibody recognition and Eu(3+)-chelate protection is prevented, allowing an efficient luminescence quenching. The assay functionality was demonstrated with a panel of EGFR inhibitors (AG-1478, compound 56, erlotinib, PD174265, and staurosporine). The monitored IC50 values ranged from 0.08 to 155.3 nM and were comparable to those found in the literature. EGFR activity and inhibition assays were performed using low nanomolar enzyme and antibody concentration in a 384-well plate format, demonstrating its compatibility for high throughput screening (HTS).
authors.affiliation: Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, 3rd Floor, FI-20520 Turku, Finland. Electronic address: natooc@utu.fi.
authors.forename: Natalia
authors.initials: N
authors.lastname: Tong-Ochoa
authors.affiliation: Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, 3rd Floor, FI-20520 Turku, Finland.
authors.forename: Kari
authors.initials: K
authors.lastname: Kopra
authors.affiliation: Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, 3rd Floor, FI-20520 Turku, Finland; Department of Biotechnology, University of Turku, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Syrjänpää
authors.affiliation: Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, 3rd Floor, FI-20520 Turku, Finland.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Legrand
authors.affiliation: Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, 3rd Floor, FI-20520 Turku, Finland.
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005063:Europium / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D049449:Luminescence; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism
chemical_list: D010455:Peptides; D014443:Tyrosine; D005063:Europium; D066246:ErbB Receptors
keywords: Epidermal growth factor receptor; High throughput screening; Quenching resonance energy transfer; Tyrosine phosphorylation