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Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins
Author(s) -
Kinoshita Eiji,
Yamada Atsushi,
Takeda Hironori,
KinoshitaKikuta Emiko,
Koike Tohru
Publication year - 2005
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200401833
Subject(s) - chemistry , affinity chromatography , agarose , chromatography , affinity electrophoresis , sepharose , phos , organic chemistry , biochemistry , enzyme
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.