Phos-tag-Based Affinity Chromatography Techniques for Enrichment of the Phosphoproteome

Abstract

Phosphorylation is among the most important post-translational modifications that regulate the function, localization, and binding specificity of particular proteins. In mammalian cells, this modification occurs mainly on serine, threonine, and tyrosine residues and is essential for the regulation of life. Changes in the phosphorylation of states of proteins fundamentally affect many cellular events and are involved in numerous diseases. Rapid and specific enrichment of native phosphoproteins from complex biological samples is therefore an important process in the fields of biology and medicine. Phosphate-affinity chromatography techniques using ions of metals such as iron, gallium, or titanium permit the effective separation of phosphorylated biomolecules, especially phosphopeptide fragments obtained by enzymatic digestion of proteins, for studies on the phosphoproteome. However, these techniques are severely limited because they cannot be applied to studies on the binding and dissociation of phosphorylated proteins at physiological pH values. This chapter introduces effective techniques for enriching phosphoproteins by using Phos-tag, a unique molecule developed to mimic the active center of an alkaline phosphatase, which can reversibly and selectively capture a phosphate monoester dianion in an aqueous solution at neutral pH values. The anion selectivity index of Phos-tag toward the phosphate dianion is at least 10,000 times higher than its selectivity toward the acetate monoanion. This characteristic of Phos-tag permits its use in the comprehensive enrichment of various biological phosphorylated compounds at physiological pH values within short time spans, providing a major advantage in obtaining information on the nature of intact native full-length phosphoproteins present in cellular lysates.