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A novel phosphate-affinity bead with immobilized Phos-tag for separation and enrichment of phosphopeptides and phosphoproteins

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Author(s): Emiko Kinoshita-Kikuta | Atsushi Yamada | Chika Inoue | Eiji Kinoshita | Tohru Koike

Journal: Journal of Integrated OMICS
ISSN 2182-0287

Volume: 1;
Issue: 1;
Start page: 157;
Date: 2011;
Original page

Keywords: Affinity chromatography | Phosphopeptide | Phosphoprotein | Phosphoproteomics | Phosphorylation | Phos-tag

ABSTRACT
A simple and efficient method was developed for separating and enriching phosphoproteins from crude biological samples containing solubil-ized cellular proteins by immobilized zinc(II) affinity chromatography. The phosphate-binding site of the affinity gel is an alkoxide-bridged dinuclear zinc(II) complex, Phos-tag, which is linked to a hydrophilic vinylic polymer bead. A novel phosphate-affinity bead (Phos-tag Toyopearl) was prepared by reaction of N-hydroxysuccinimide-activated Toyopearl AF-Carboxy-650M gel with a 2-aminoethylcarbamoyl de-rivative of Phos-tag. Phosphopeptides were retrieved quantitatively and selectively on a ┬ÁL-scale column at room temperature. The column was stable for long-term storage and could be reused many times. The technique was used to separate and enrich phosphoproteins from an epider-mal growth factor-stimulated human epidermoid carcinoma A431 cell lysate. The operations necessary for 1-mL-scale open-column chroma-tography were conducted at a physiological pH during 1 h. The strong enrichment of the phosphoproteins into the eluted fraction was evaluat-ed by gel electrophoresis, followed by Western blotting with Phos-tag Biotin and several antibodies, Pro-Q Diamond phosphoprotein gel stain-ing, and mass spectrometry.
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