Advances in Phos-tag-based methodologies for separation and detection of the phosphoproteome.

Abstract

This review article describes analytical techniques based on the phosphate-binding tag molecule "Phos-tag", which is an alkoxide-bridged dinuclear metal complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate, for studying the protein phosphorylome. The dinuclear zinc(II) complex forms a stable 1:1 complex with a phosphate monoester dianion in an aqueous solution under conditions of neutral pH. By using a series of functional Phos-tag derivatives, our group has developed novel techniques that are useful in studies on kinomics and phosphoproteomics. Among the derivatives, a series of biotinylated Phos-tag derivatives have been used as molecular tools in applications such as Western blotting for comprehensive detection of phosphorylated proteins and in highly sensitive peptide microarray-based techniques for the detection of kinase activities in biological samples. The review also gives an outline of phosphate affinity electrophoresis, in which immobilized Phos-tag molecules in a general polyacrylamide gel are used to separate proteins and detect differences in their phosphorylation status. This technique permits quantitative analyses of multiple phosphorylation statuses of individual cellular proteins and their time-dependent changes. Conventional mass spectrometry-based shotgun techniques used in phosphoproteomics detect the phosphorylation modification of proteins in peptide fragments, whereas the Phos-tag electrophoresis technique permits the direct analysis of the phosphorylation status of full-length proteins. The technique therefore provides a greater understanding of the detailed properties of particular proteins involved in specific physiological and pathological events. This article is part of a Special Issue entitled: Medical Proteomics.