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Molecular & Cellular Proteomics 6:1656-1665, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis^*,S

Shun Feng^,,¶, Mingliang Ye^,¶, Houjiang Zhou, Xiaogang Jiang, Xingning Jiang, Hanfa Zou^,|| and Bolin Gong^**

From the National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, China, College of Chemistry and Chemical Engineering, Xinjiang University, Urumqi, Xinjiang 830046, China, and ^** Key Laboratory of Biotechnology, Ningxia University, Yin chuan 750021, China

Large scale characterization of phosphoproteins requires highly specific methods for purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. Enrichment of phosphopeptides from complex peptide mixtures by IMAC is a popular way to perform phosphoproteome analysis. However, conventional IMAC adsorbents with iminodiacetic acid as the chelating group to immobilize Fe³⁺ lack enough specificity for efficient phosphoproteome analysis. Here we report a novel IMAC adsorbent through Zr⁴⁺ chelation to the phosphonate-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads. The high specificity of Zr⁴⁺-IMAC adsorbent was demonstrated by effectively enriching phosphopeptides from the digest mixture of phosphoprotein (- or ß-casein) and bovine serum albumin with molar ratio at 1:100. Zr⁴⁺-IMAC adsorbent was also successfully applied for the analysis of mouse liver phosphoproteome, resulting in the identification of 153 phosphopeptides (163 phosphorylation sites) from 133 proteins in mouse liver lysate. Significantly more phosphopeptides were identified than by the conventional Fe³⁺-IMAC approach, indicating the excellent performance of the Zr⁴⁺-IMAC approach. The high specificity of Zr⁴⁺-IMAC adsorbent was found to mainly result from the strong interaction between chelating Zr⁴⁺ and phosphate group on phosphopeptides. Enrichment of phosphopeptides by Zr⁴⁺-IMAC provides a powerful approach for large scale phosphoproteome analysis.

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