A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules

• We report a novel magnetic bead for phosphate-affinity separation.
• The target molecules are phosphomonoester-type nucleotides and phosphopeptides.
• The separation can be conducted by using aqueous solutions at neutral pH.
• The operation time from sample addition to elution is less than 12 min.
• The phosphate-affinity beads are stable for multiple use and long-term storage.

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 μL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol.