Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis

Shun Feng; Mingliang Ye; Houjiang Zhou; Xiaogang Jiang; Xingning Jiang; Hanfa Zou; Bolin Gong

doi:10.1074/mcp.T600071-MCP200

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

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

‖To whom correspondence should be addressed. Tel.: 86-411-84379620; Fax: 86-411-84379610; E-mail: hanfazou{at}dicp.ac.cn

Abstract

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 (α- or β-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.

Footnotes

Published, MCP Papers in Press, June 17, 2007, DOI 10.1074/mcp.T600071-MCP200
↵ ¹ The abbreviations used are: GMA-EDMA, poly(glycidyl methacrylate-co-ethylene dimethacrylate); Xcorr, cross-correlation value; ΔCn, delta Cn value.
↵ ² The raw data for the three LC-MS/MS runs obtained by the LTQ are available upon request.
↵* This work was supported by National Natural Sciences Foundation of China Grant 20675081, China State Key Basic Research Program Grant 2005CB522701, China High Technology Research Program Grant 2006AA02A309, the Knowledge Innovation program of the Chinese Academy of Sciences (Grant KJCX2.YW.HO9) and the Knowledge Innovation program of the Dalian Institute of Chemical Physics (to H. Z.), and National Natural Sciences Foundation of China Grant 20605022 (to M. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material (tables and the labeled spectra for identified phosphopeptides).
↵¶ Both authors contributed equally to this work.
- Received December 28, 2006.
- Revision received May 9, 2007.