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首页> 外文期刊>Molecular & cellular proteomics: MCP >Identification of Metal-binding Proteins in Human Hepatoma Lines by Immobilized Metal Affinity Chromatography and Mass Spectrometry
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Identification of Metal-binding Proteins in Human Hepatoma Lines by Immobilized Metal Affinity Chromatography and Mass Spectrometry

机译：固定化金属亲和色谱和质谱法鉴定人肝癌细胞系中的金属结合蛋白

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摘要

The metalloproteome is defined as the set of proteins that have metal-binding capacity by being metalloproteins or having metal-binding sites. A different metalloproteome may exist for each metal. Mass spectrometric characterization of metalloproteomes provides valuable information relating to cellular disposition of metals physiologically and in metal-associated diseases. We examined the Cu and Zn metalloproteomes in three human hepatoma lines: Hep G2 and Mz-Hep-1, which retain many functional characteristics of normal human hepatocytes, and SK-Hep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep G2 cells depleted in vitro of cellular copper. We used matrix-assisted laser desorption ionization and electrospray ionization quadrupole time-of-flight mass spectrometry to analyze peptide sequences of tryptic digests obtained by either in-gel digestion of metal-binding proteins or peptides on an immobilized metal affinity chromatography column loaded with either Cu or Zn. Mainly high abundance proteins were identified. Cu-binding proteins identified included enolase, albumin, transferrin, and alcohol dehydrogenase as well as certain intracellular chaperone proteins. The Cu metalloproteome was not identical to the Zn metalloproteome. Peptide binding experiments demonstrated that Cu coordination prefers the order of residues histidine > methionine > cysteine. Although the Cu metalloproteome was similar from line to line, subtle differences were apparent. Gel profiling showed more extensive variation in expression of annexin II in SK-Hep-1 and Mz-Hep-1 than in Hep G2 and normal liver tissue. Glycerylphosphorylethanolamine was identified as a post-translational modification at residue Glu-301 of elongation factor 1-α in Hep G2. Intracellular copper depletion was associated with loss of the glycerylphosphoryl side group. These findings suggest that post-translational modification could be affected by intracellular actions of copper. Comparison of the Cu and Zn metalloproteomes in Hep G2 with a published general proteome of Hep G2 disclosed little overlap (Seow, T. K., et al. (2001) Proteomics 1, 1249-1263). Proteins in the metalloproteomes of human hepatocytes can be identified by these methods. Variations in these metalloproteomes may have important physiological relevance.
机译：金属蛋白质组定义为通过成为金属蛋白或具有金属结合位点而具有金属结合能力的一组蛋白质。每种金属可能存在不同的金属蛋白质组。金属蛋白质组的质谱表征提供了有关生理上以及与金属相关的疾病中金属细胞处置的有价值的信息。我们检查了三种人类肝癌细胞系中的铜和锌金属蛋白质组：Hep G2和Mz-Hep-1，它们保留了正常人类肝细胞的许多功能特征，而SK-Hep-1，它们的分化程度很低。另外，我们研究了正常人肝脏和肝铜细胞中耗尽的Hep G2细胞的单个标本。我们使用基质辅助激光解吸电离和电喷雾电离四极杆飞行时间质谱仪分析了胰蛋白酶消化物的肽序列，该胰蛋白酶消化物是通过金属结合蛋白或肽的凝胶消化在固定的载有金属亲和色谱柱上获得的铜或锌。主要鉴定出高丰度蛋白。鉴定出的Cu结合蛋白包括烯醇酶，白蛋白，转铁蛋白和醇脱氢酶，以及某些细胞内伴侣蛋白。铜金属蛋白质组与锌金属蛋白质组不相同。肽结合实验表明，Cu配位优选组氨酸>蛋氨酸>半胱氨酸的残基顺序。尽管各行的铜金属蛋白质组相似，但细微的差异是显而易见的。凝胶谱分析显示，与Hep G2和正常肝组织相比，SK-Hep-1和Mz-Hep-1中膜联蛋白II的表达变化更大。甘油基磷酸基乙醇胺被鉴定为Hep G2中延伸因子1-α的残基Glu-301的翻译后修饰。细胞内铜耗竭与甘油基磷酰基侧基的损失有关。这些发现表明翻译后修饰可能受到铜的细胞内作用的影响。比较Hep G2中的Cu和Zn金属蛋白质组与已发布的Hep G2通用蛋白质组相比，几乎没有重叠（Seow，T. K.等人（2001）Proteomics 1，1249-1263）。可以通过这些方法鉴定人肝细胞的金属蛋白质组中的蛋白质。这些金属蛋白质组的变异可能具有重要的生理相关性。

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