リン酸化プロテオミクスのためのPhos-tagテクノロジー

Phos-tag Technology for Phosphoproteomics

木下 英司

木下 恵美子

小池 透

分析化学

Phos-tagは,中性の水溶液中においてリン酸モノエステルジアニオンを選択的に捕捉する機能性分子である.Phos-tagのリン酸モノエステルジアニオンに対する親和性は,生体中に存在する他のアニオン,例えばカルボン酸アニオンに対するそれよりも1万倍以上大きい.著者らは,Phos-tagを基盤としたリン酸化プロテオミクスに有用な技術(Phos-tagテクノロジー)を開発し,実用化している.本稿では,これまでに実用化した三つの技術について概説する.一つ目は,Phos-tagアクリルアミドを用いた電気泳動によってリン酸化タンパク質を分離する技術,二つ目はビオチン化Phos-tagを用いてウェスタンブロット膜上に転写されたリン酸化タンパク質を検出する技術,三つ目はPhos-tagアガロース,あるいはPhos-tagトヨパールを用いてリン酸化タンパク質を分離・濃縮するクロマトグラフィー技術である.

Reversible protein phosphorylation is a key signaling mechanism for modulating the functional properties of proteins in various cellular processes. More than 500 protein kinases and more than 100 protein phosphatases are known or predicted in the human proteome alone. The numbers clearly reflect the importance of protein phosphorylation. In fact, abnormal phosphorylation by perturbation of the balance of these enzyme reactions is deeply related to a wide range of human diseases, including cancer, diabetes mellitus, neurodegeneration, and immune/inflammatory disorders. Methods for the determination of the phosphorylation status of a certain protein are thus very important with respect to evaluating the basis for understanding the molecular origins of diseases, and for drug design. Recently, we found that a dinuclear metal complex (Phos-tag) of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acts as a novel phosphate-binding tag molecule in an aqueous solution under physiological conditions. Phos-tag has a vacancy on two metal ions, which is suitable for the access of a phosphomonoester dianion (R-OPO3_3^<2->) as a bridging ligand. A dinuclear zinc(II) complex (Zn^<2+>-Phos-tag) strongly binds to phenyl phosphate dianion (Kd=2.5×10^<-8> M) at a neutral pH. The anion selectivity indexes against SO_4^2-, CH_3COO^-, Cl^-, and the bisphenyl phosphate monoanion at 25 ℃ are 5.2×10^3, 1.6×10^4, 8.0×10^5, and>2×10^6,respectively. By utilizing the Phos-tag molecule and its derivatives, we developed three major Phos-tag technologies, and put them into practical use for a phosphoproteome study. Herein, we describe convenient and reliable methods for the detection of phosphorylated proteins, such as affinity electrophoresis using Phos-tag acrylamide, Western blotting using biotinylated Phos-tag, and affinity chromatography using Phos-tag agarose or Phos-tag TOYOPEARL.

affinity chromatography

affinity electrophoresis

phosphoproteomics

Phos-tag

Western blotting

化学 [ 430 ]

日本語

日本分析化学会

Copyright (c) 2012 The Japan Society for Analytical Chemistry

本文データはJ-STAGEから複製したものである

[ISSN] 0525-1931

[DOI] 10.2116/bunsekikagaku.61.469

[NCID] AN00222633

[DOI] http://dx.doi.org/10.2116/bunsekikagaku.61.469