A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides

Takiyama Kei

Kinoshita Eiji

Kinoshita Emiko

Fujioka Yoshitake

Kubo Yusuke

Koike Tohru

Analytical Biochemistry

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here, we introduce a novel FRET system for the detection of phosphopeptides using a phosphate-binding tag molecule, Zn2+-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). Carboxyfluorescein (FAM)-labeled phospho- and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, em 520 nm) and AMCA (a fluorescent donor, ex 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn2+-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 dephosphorylation of the FAM-labeled protein-tyrosine phosphatase 1B substrate

FRET

Phos-tag

Phosphorylation

Dephosphorylation

Phosphopeptide

Biology [ 460 ]

eng

Elsevier

Copyright (c) 2009 Elsevier Inc.

[ISSN] 0003-2697

[ISSN] 1096-0309

[DOI] 10.1016/j.ab.2009.02.039

[NCID] AA00524867

[NCID] AA11537838

[DOI] http://dx.doi.org/10.1016/j.ab.2009.02.039