邹汉法—磷酸化蛋白质组学研究新成果

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生物谷报道：中科院大连化物所生物技术部1809组封顺、叶明亮、邹汉法等人关于新型金属离子固定化亲和色谱固定相应用于磷酸化蛋白质组学的研究成果(Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphro proteome Analysis)发表于9月份出版的《分子细胞蛋白质组学》(Molecular & Cellular Proteomics,MCP,2007，6，1656-1665）。《分子细胞蛋白质组学》由美国生物化学和细胞生物学学会主办，是蛋白质组学研究领域最高影响因子的学术刊物，2005年和2006年的SCI引用影响因子分别为9.876和9.62。

在复杂的蛋白质提取物的酶解产物中，磷酸化多肽的高选择性、高稳定性富集是磷酸化蛋白质组学分析的关键步骤之一。常规的金属离子固定化亲和色谱固定相以氨基二乙酸(IDA)或次氮基三乙酸(NTA)为螯合基团与Fe3+，Ga3+等离子螯合，进而选择性地作用于磷酸化肽段，达到富集磷酸化多肽的目的。这一类吸附材料对含有酸性氨基酸残基侧链的肽段也具有富集作用，因此严重干扰磷酸化肽段的检测。本项工作制备了磷酸脂改性的高分子材料微球，利用磷酸脂基团与镐离子的配位作用固定化镐离子，再通过镐离子与磷酸肽上的磷酸基团的选择性作用达到富集的目标。实验结果表明镐离子固定化亲和色谱固定相对磷酸化肽段具有很高选择性和富集效果。（援引中科院大连化物所）

原始出处:

Molecular & Cellular Proteomics 6:1656-1665, 2007.

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

Shun Feng ${ddagger}$ , $§$ ,¶, Mingliang Ye ${ddagger}$ ,¶, Houjiang Zhou ${ddagger}$ , Xiaogang Jiang ${ddagger}$ , Xingning Jiang ${ddagger}$ , Hanfa Zou ${ddagger}$ ,|| and Bolin Gong**

From the ${ddagger}$ 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 ( ${alpha}$ - 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.