Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins |
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Authors: | Kinoshita Eiji Yamada Atsushi Takeda Hironori Kinoshita-Kikuta Emiko Koike Tohru |
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Institution: | Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan. kinoeiji@hiroshima-u.ac.jp |
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Abstract: | 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-bisbis(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 CH3CN. 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, alpha(s1)-casein, and beta-casein at physiological pH. |
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Keywords: | Affinity chromatography Zinc(II) complex Phospho‐proteomics Phosphopeptide Phosphorylated protein |
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