Immobilized metal affinity chromatography of phosphorylated proteins using high performance sorbents

The interactions of two model phosphoproteins (porcine pepsin and ovalbumin) with two different immobilized metal affinity chromatography (IMAC) sorbents containing immobilized Fe(3+), Ga(3+), and UO(2)(2+) ions have been investigated under various conditions. Both proteins were adsorbed on immobilized uranyl ions under acidic conditions similar to those on immobilized Fe(3+) and Ga(3+) ions. The retained proteins could be released either by the presence of phosphate ions in the elution buffer (immobilized Ga(3+) and Fe(3+) ions) or by an increased pH (all tested immobilized ions). The IMAC sorbents employed could be used under the conditions of high-performance chromatography and are suitable for the separation and analysis of intact phosphoproteins.