Zr(IV)-immobilized Affinity Beads Prepared by Surface Template Polymerization for Capturing Phosphorylated Proteins
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Zr(IV)-immobilized affinity beads prepared by surface template polymerization for capturing phosphorylated proteins Kazuya Uezu, Hidenobu Mizuki, Yudai Ito, Hisashi Harada and Haruka Oshiumi Life and Environment Engineering, The University of Kitakyushu, Kitakyushu, Fukuoka, Japan ABSTRACT A novel immobilized metal affinity chromatography (IMAC) bead, Zr(IV)-immobilized resin, was prepared by surface template polymerization to enrich phosphorylated proteins and peptides from complex peptides mixtures. In order to enhance both the kinetics and the efficiency, large pathways for the proteins and peptides in the resin were formed, and the Zr(IV)phosphate complexes were immobilized on the polymer surface. The morphology of the Zr(IV)immobilized resin was evaluated the by measuring the specific surface area, pore volume, and pore distribution. The resin possessed large amount of the large-macro pores around 300 nm. The separation performance of β-casein from bovine serum albumin (BSA) solution was evaluated by phosphopeptide enrichment and MALDI-TOF MS analysis. The Zr(IV)-immobilized resin showed the high selectivity of the phosphopeptide.
INTRODUCTION The development of new efficient methods for highly specific enrichment of phosphorylated proteins and peptides is one of the most active research fields in phosphorproteome analysis. Enrichment of phosphorylated proteins and peptides from complex peptides mixtures by immobilized metal affinity chromatography (IMAC) is a popular way to perform phosphoproteome analysis. IMAC is originally based on the affinity of the phosphate group with metal ions (usually Fe(III) or Ga(III)) immobilized on a choromatographic support. However, the specificity of those IMAC adsorbents is still not high enough [1]. To characterize phosphoproteins more efficiently, it was necessary to develop novel IMAC beads with higher affinity surface for phosphoproteins. In this study, we focused on the strong interaction between zirconium phosphonate (Zr-Phos complex) and phosphate groups [2]. Taking advantage of strong interaction, we have developed a novel IMAC adsorbent immobilized Zr(IV) on the polymer surface (Zr-Phos IMAC) by surface template polymerization with W/O/W emulsion. The surface template polymerization method using a functional host monomer, an emulsion stabilizer, a polymer matrix co-monomer, and a target molecule (ion) should be promising for the preparation of such resins because it is possible to immobilize the recognition site on the polymer surface [3-7]. Phosphoric acid oleyl ester (DOLPA), sorbitan monooleate (Span80), divinyl-benzene (DVB), polystyrene, and toluene were employed as the functional monomer, emulsion stabilizer, matrix-forming monomer, porogen, and diluent, respectively. β-casein was employed as a model phosphoprotein to investigate the performance of a novel Zr-Phos IMAC. The separation performance of β-casein from bovine serum albumin (BSA) solution was evaluated by phosphopeptide enrichment and MALDI-TOF MS analysis.
Fig.1 Zr(IV)-immobilized af
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