Knowledge-based proteome profiling:: Considering identified proteins to evaluate separation efficiency by 2-D PAGE
Knowledge-based proteome profiling:: Considering identified proteins to evaluate separation efficiency by 2-D PAGE
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DOI：
10.1002/elps.200500964
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发表时间：
2006-07-01
期刊：
影响因子：
2.9
通讯作者：
Gerner, Christopher
中科院分区：
文献类型：
--
作者：
Gundacker, Nina;Bayer, Editha;Gerner, Christopher
蛋白质组分析技术依赖于蛋白质或肽的分离及其随后的定量。该技术的可靠性仍然有限，因为蛋白质组分析结果不一定代表分析样品的真实蛋白质组成，从而严重妨碍了正确的数据解释。许多实验观察到的蛋白质组改变在生物学上并不显着。本研究的目的是利用蛋白质生物学背景的知识来建立优化的蛋白质组分析方案。虽然发现总细胞蛋白级分的二维斑点图案不能很好地代表真实的蛋白质组成，但发现纯化的亚细胞蛋白级分更好地代表了分析样品的蛋白质组成。将标准化方案应用于不同种类的细胞揭示了一些惊人的观察结果。首先，不同来源的培养细胞的蛋白质组成非常相似。其次，从生物学的角度来看，用所描述的方案观察到的蛋白质组改变确实有意义，因此可以被认为是分析样品的真正代表。第三，从不同供体分离的原代白细胞被发现表现出微小但可重复且显着的个体差异。我们将蛋白质组谱中已识别蛋白质的已知特性的考虑称为基于知识的方法。目前的数据表明，这种方法可能极大地有助于改进应用技术和评估结果。我们证明，满足明确的蛋白质组图谱标准最终会产生可靠且生物学相关的数据。
Proteome profiling techniques rely on the separation of proteins or peptides and their subsequent quantification. The reliability of this technique is still limited because a proteome profiling result does not necessarily represent the true protein composition of the analysed sample, thus seriously hampering proper data interpretation. Many experimentally observed proteome alterations are biologically not significant. It was the aim of this study to use the knowledge of the biological context of proteins in order to establish optimised proteome profiling protocols. While 2-D spot patterns of total cell protein fractions were found to poorly represent the true protein composition, purified subcellular protein fractions were found to better represent the protein composition of the analysed sample. The application of a standardised protocol to different kinds of cells revealed several striking observations. Firstly, the protein composition of cultured cells of various origins is very similar. Secondly, proteome alterations observed with the described protocols do make sense from a biologic point of view and may thus be considered as truly representative for the analysed samples. Thirdly, primary white blood cells isolated from different donors were found to show minor, but reproducible and significant individual differences. We designate the consideration of known properties of identified proteins in proteome profiles as a knowledge-based approach. The present data suggest that this approach may tremendously help to improve the applied techniques and assess the results. We demonstrate that the fulfilment of well-defined criteria of proteome profiles eventually results in reliable and biologically relevant data.