Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins

Immobilized metal ion affinity chromatography (IMAC) is now a widely accepted technique for the separation of natural or artificial products that is beginning to find industrial applications. Here, we introduce a novel procedure for the separation of phosphopeptides and phosphorylated proteins by immobilized zinc(II) affinity chromatography. The phosphate‐binding site of the affinity gel is an alkoxide‐bridged dinuclear zinc(II) complex, the 1,3‐bis[bis(pyridin‐2‐ylmethyl)amino]propan‐2‐olato dizinc(II) complex ( Phos‐tag ), which is linked to a highly cross‐linked 4% ( w/v ) agarose. The affinity gel ( Phos‐tag agarose ) was prepared by the quantitative reaction of N ‐hydroxysuccinimide‐activated Sepharose and a Phos‐tag derivative having a 2‐aminoethylcarbamoyl group in dry CH 3 CN. Phosphopeptides were retrieved in a quantitative and highly selective manner by a spin column method using Phos‐tag agarose at room temperature. Furthermore, in this study, we demonstrate a simple, rapid, and reusable affinity column chromatography for the separation of phosphorylated proteins such as ovalbumin, α s1 ‐casein, and β‐casein at physiological pH.