Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis
Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis
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DOI：
10.1111/j.1742-4658.2007.05705.x
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发表时间：
2007-03-01
期刊：
影响因子：
5.4
通讯作者：
Hattori, Seisuke
中科院分区：
文献类型：
--
作者：
Machida, Mitsuyo;Kosako, Hidetaka;Hattori, Seisuke
在蛋白质组学分析中，预折射程序有利于低丰度蛋白质的鉴定。在这里，我们已经优化了固定化金属亲和色谱（IMAC）富集磷蛋白的条件。金属离子，Ga（III），Fe（III），Zn（II）和Al（III），被比较了它们捕获磷蛋白的能力; Ga（III）是最好的。IMAC的pH值和离子强度的详细分析使我们能够确定最佳条件（pH 5.5和0.5 m NaCl）。当全细胞裂解物以这种方式分级分离时，约十分之一的总蛋白质被回收到细胞内，磷酸化的细胞外信号调节激酶（ERK）的回收率超过90%。磷酸化形式的核糖体S6激酶（RSK）和Akt也富集在相同的条件下有效。我们的Ga（III）IMAC和市售的磷蛋白纯化试剂盒的表现相似，磷蛋白的光谱略有差异。当磷酸化蛋白富集从NIH 3 T3细胞中，ERK被激活或抑制进行了分析，通过二维荧光差异凝胶电泳，磷酸化ERK被检测为离散的斑点独特的ERK激活的细胞，与周围的斑点重叠的情况下，prefractionation。我们应用相同的技术来寻找Akt底物，并将Abelson interactor 1确定为新的潜在靶点。这些结果证明了IMAC富集磷蛋白的有效性，并表明这种方法将在磷蛋白质组研究中普遍使用。
Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.