Development and Application of Phos-tag Technology for Phosphoproteomics

Abstract

We found that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acted as a novel phosphate-binding tag nanomolecule, Phos-tag, in an aqueous solution under near physiological conditions. The metal complex having a vacancy on two metal ions 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 a p-nitrophenyl phosphate dianion (K_d = 2.5 × 10⁻⁸ M) at a neutral pH. The anion selectivity indexes against SO₄²⁻, CH₃COO⁻, Cl⁻, and the bisphenyl phosphate monoanion at 25°C are 5.2 × 10³, 1.6 × 10⁴, 8.0 × 10⁵, and >2 × 10⁶, respectively. The author has been involved in developing a technology by using the Phos-tag molecule and its derivatives to permit the analysis of phosphorylated biomolecules. To date, Phos-tag technology has contributed to the development of several procedures for phosphoproteomics, including a phosphate-affinity chromatography technique for the separation and enrichment of phosphopeptides and phosphoproteins, a wide variety of microarray/on-chip techniques for the detection of protein phosphorylation multiplexes, and a phosphate-affinity electrophoresis technique for the detection of shifts in the mobilities of phosphoproteins. In this article, the author introduces the impact of Phos-tag-based technological advances for phosphoproteomics.