Circumventing the problems caused by protein diversity in microarrays: Implications for protein interaction networks
Circumventing the problems caused by protein diversity in microarrays: Implications for protein interaction networks
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DOI：
10.1021/ja065381g
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发表时间：
2006-10-25
影响因子：
15
通讯作者：
MacBeath, Gavin
中科院分区：
文献类型：
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作者：
Gordus, Andrew;MacBeath, Gavin
蛋白质微阵列提供了一种控制良好的高通量方法来揭示蛋白质-蛋白质相互作用。然而，这种和其他标准化测定的一个问题是，蛋白质在其物理性质方面变化很大。如果使用简单的基于阈值的方法来定义蛋白质-蛋白质相互作用，则所得的二元网络可能具有很强的偏差。在这里，我们调查的程度，即使是密切相关的蛋白质相互作用域的变化时，打印成微阵列。我们发现，当一组表现良好的单体Src同源2（SH 2）结构域以相同的浓度打印时，它们相对于所得活性蛋白的表面密度变化高达50倍。当使用荧光标记的磷酸肽对这些结构域进行基于阈值的结合测定时，出现了潜在的生物物理相互作用的误导性图片。然而，这个问题可以通过获得每个蛋白质-肽相互作用的饱和结合曲线来解决。重要的是，从这些曲线获得的平衡解离常数是独立的活性蛋白质的表面密度。我们认为，应该更加重视从蛋白质微阵列中获得定量信息，这应该作为一个更普遍的目标，在所有的努力，以确定大规模的蛋白质相互作用网络。
Protein microarrays provide a well-controlled, high-throughput way to uncover protein−protein interactions. One problem with this and other standardized assays, however, is that proteins vary considerably with respect to their physical properties. If a simple threshold-based approach is used to define protein−protein interactions, the resulting binary networks can be strongly biased. Here, we investigate the extent to which even closely related protein interaction domains vary when printed as microarrays. We find that, when a collection of well behaved, monomeric Src homology 2 (SH2) domains are printed at the same concentration, they vary by up to 50-fold with respect to the resulting surface density of active protein. When a threshold-based binding assay is performed on these domains using fluorescently labeled phosphopeptides, a misleading picture of the underlying biophysical interactions emerges. This problem can be circumvented, however, by obtaining saturation binding curves for each protein−peptide interaction. Importantly, the equilibrium dissociation constants obtained from these curves are independent of the surface density of active protein. We submit that an increased emphasis should be placed on obtaining quantitative information from protein microarrays and that this should serve as a more general goal in all efforts to define large-scale protein interaction networks.