A FRET-based Protein Kinase Assay Using Phos-tag-modified Quantum Dots and Fluorophore-labeled Peptides

We have developed a novel FRET-based assay to monitor protein kinase activity using quantum dots (QDs) and fluorophore-labeled substrate peptides. To develop a FRET-based protein kinase assay, it is important to consider the phosphate group recognition strategy and to ensure that the FRET pairs are close enough because the FRET efficiency is highly dependent on the distance between the FRET pairs. Here, we incorporated a phos-tag, which captures phosphate groups strongly and selectively, into a protein kinase assay to recognize phosphorylation. Our detection system was composed of phos-tag-modified QDs and Cy5-labeled substrate peptides. Because the phos-tags capture phosphate groups by forming dinuclear complexes, the Cy5-labeled substrate peptides are captured by the phos-tags on the QD surface upon protein kinase-mediated phosphorylation, which induces FRET from the QDs to Cy5 because of the approximation of Cy5 to the QDs. On the basis of the difference of this FRET efficiency, we successfully measured protein kinase A activity, which demonstrated the feasibility of our assay.