While phosphoproteins have attracted great interest toward the post-genome research (e.g., clinical diagnosis and drug design), there have been few procedures for the specific separation and enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient procedure to enrich phosphorylated proteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a dinuclear zinc(II) complex, Phos-tag, attached on a hydrophilic vinyl-polymeric bead (TOYOPEARL, Tosoh Corp., Tokyo, Japan). TOYOPEARL is more excellent than other column matrix beads, such as agarose, in physical and chemical properties. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 0.50 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated proteins from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 0.50 M NaCl. The concentrations of CH3COONa and NaCl used are only half relative to the conventional protocol of phosphate-affinity chromatography using Phos-tag Agarose. In this study, we demonstrate the separation and enrichment of phosphoproteins from an EGF-stimulated human epidermoid carcinoma A431 cell lysate using the novel phosphate-affinity bead and improved buffer conditions. The total time for the column chromatography (1 mL-beads scale) was less than one hour. The strong enrichment of the phosphorylated proteins into the elution fraction was evaluated using SDS-PAGE and 2-DE (IEF/SDS-PAGE) followed by Western blotting with a biotin-pendant Phos-tag and some antibodies, Pro-Q Diamond phosphoprotein gel staining, and MS analyses.
