History of Phos-tag technology for phosphoproteomics

Phos-tag is a functional molecule that selectively captures a phosphate monoester dianion in neutral aqueous solutions. The affinity of Phos-tag for phosphate monoester dianions is more than 10,000 times greater than that for other anions present in living organisms, such as carboxylic acid anions. We have developed and applied useful techniques for phosphoproteomics based on Phos-tag. This review describes the history of Phos-tag development and outlines three main technologies that have been put to practical use. The first is a technique to separate and concentrate phosphopeptides and phosphoproteins using a Phos-tag derivative with a hydrophilic chromatography carrier (Phos-tag polymer beads). The second is a technology to detect phosphopeptides and phosphoproteins on various arrays using Phos-tag biotin. The third is a technique to separate and detect phosphoproteins by electrophoresis using Phos-tag acrylamide. We hope that these three technologies will make a significant contribution to phosphoproteomics and, ultimately, to life science research. SIGNIFICANCE: The authors 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-OPO₃^2-) 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 SO₄^2-, CH₃COO^-, Cl^-, and the bisphenyl phosphate monoanion at 25 °C are 5.2 × 10³, 1.6 × 10⁴, 8.0 × 10⁵, and > 2 × 10⁶, respectively. We have been involved in developing technologies 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, and a phosphate-affinity electrophoresis technique for the detection of shifts in the mobilities of phosphoproteins. In this review article, the authors introduce the impact of Phos-tag-based technological advances for phosphoproteomics.