We introduce a novel fluorescence resonance energy transfer (FRET) system for the detection of a phosphorylated molecule such as a phosphopeptide using a phosphate-binding tag molecule, Zn(II)-Phos-tag (1,3-bis[bis (pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) attached with a 7-amino-4-methylcoumarin-3-acetic acid (AMCA). 5-Carboxyfluorescein (FAM)-labeled phosphopeptides and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, emission at 520 nm) and AMCA (a fluorescent donor, excitation at 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn(II)-Phos-tag captured specifically the FAM-labeled phosphopeptide to form a stable 1:1 complex, resulting in efficient FRET. After the FAM-labeled phosphopeptide was dephosphorylated with alkaline phosphatase, the FRET disappeared. Using this FRET system, we demonstrated the detection of the time-dependent reversible phosphorylation of the FAM-labeled substrate peptide. The Phos-tag-based FRET system has the following major advantages: i) The real-time analysis of the reversible phosphorylation reaction is possible without multiple samplings, ii) the analysis requires a simple procedure just using two solutions of AMCA-labeled Phos-tag and a FAM-labeled compound, and iii) the system would be useful for the reliable and comprehensive phosphorylation assays for various phosphopeptides containing phosphoserine, phosphothreonine, or phosphotyrosine, in vitro
. Thus, the principle of this system would be applied to high-throughput kinase/phosphatase profiling, measurement of enzyme activity, and determination of an activator or an inhibitor.