A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides
A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides
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DOI：
10.1016/j.ab.2009.02.039
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发表时间：
2009-05-15
影响因子：
2.9
通讯作者：
Koike, Tohru
中科院分区：
文献类型：
--
作者：
Takiyama, Kei;Kinoshita, Eiji;Koike, Tohru
荧光共振能量转移（FRET）是两个染料分子的电子激发态之间的距离依赖性相互作用。在这里，我们介绍了一种新的FRET系统检测磷酸肽使用的磷酸结合标签分子，锌2 +-Phos-标签（1，3-双[双（吡啶-2-基甲基）氨基]丙烷-2-羟基合二锌（II）配合物）连接7-氨基-4-甲基香豆素-3-乙酸（AMCA）。制备了羧基荧光素（FAM）标记的磷酸肽和非磷酸肽作为FRET系统的靶分子。一组FAM（荧光受体，λ（ex）520 nm）和AMCA（荧光供体，λ（ex）345 nm）经常用于FRET系统。AMCA标记的Zn ~（2+）-Phos标记物特异性地捕获FAM标记的磷酸肽，形成稳定的1：1复合物，导致高效的FRET。FAM标记的磷酸肽用碱性磷酸酶去磷酸化后，FRET消失。使用该FRET系统，我们证明了FAM标记的蛋白酪氨酸磷酸酶1B底物的时间依赖性去磷酸化的检测。(c)2009年EIsevier Inc. All rights reserved.
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(pytidin-2-ylmethy)aminio]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, lambda(ex) 520 nm) and AMCA (a fluorescent donor, lambda(ex) 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn2+-Phos-tag, specifically captured 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. (c) 2009 EIsevier Inc. All rights reserved.