Phos-tag Technology for Phosphoproteomics

作者：Kinoshita Eiji^*; Kinoshita Kikuta Emiko; Koike Tohru

来源：Bunseki Kagaku, 2012, 61(6): 469-487.

DOI：10.2116/bunsekikagaku.61.469

• affinity chromatography
• affinity electrophoresis
• phosphoproteomics
• Phos-tag
• Western blotting

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-OPO32-) as a bridging ligand. A dinuclear zinc(II) complex (Zn2+ - Phos-tag) strongly binds to phenyl phosphate dianion (K-d = 2.5 x 10(-8) M) at a neutral pH. The anion selectivity indexes against SO42-, CH3COO-, Cl-, and the bisphenyl phosphate monoanion at 25 degrees C are 5.2 x 10(3), 1.6 x 10(4), 8.0 x 10(5), and %26gt; 2 x 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.

• 出版日期2012-6