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 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.
Kinoshita
Eiji
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan. kinoeiji@hiroshima-u.ac.jp
Yamada
Atsushi
A
Takeda
Hironori
H
Kinoshita-Kikuta
Emiko
E
Koike
Tohru
T
D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008024:Ligands; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D011506:Proteins / Q000032:analysis; D012685:Sepharose / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays; D015032:Zinc / Q000737:chemistry*
D002364:Caseins; D003432:Cross-Linking Reagents; D007477:Ions; D008024:Ligands; D010455:Peptides; D010748:Phosphopeptides; D011506:Proteins; D012685:Sepharose; D014357:Trypsin; D015032:Zinc