Protein Complex Identification and quantitative complexome by CN-PAGE
Protein Complex Identification and quantitative complexome by CN-PAGE
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DOI：
10.1038/s41598-019-47829-7
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发表时间：
2019-08-08
影响因子：
4.6
通讯作者：
Graf, Alexander
中科院分区：
文献类型：
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作者：
Gorka, Michal;Swart, Corne;Graf, Alexander
大多数细胞过程是由蛋白质复合物完成的。各种大小的分馏方法以前已与质谱相结合，以确定蛋白质复合物。然而，这些方法大多缺乏定量信息，而定量信息是了解蛋白质复合物丰度和组成变化如何影响代谢通量所必需的。在本文中，我们提出了一种概念验证方法来定量研究模式植物拟南芥（Arabidopsis thaliana）在白天结束（ED）和晚上结束（EN）时的复合体。我们发现，通过Clear-Native-PAGE （CN-PAGE）对天然蛋白复合物进行大小分级，结合质谱法可以沿分子量梯度建立丰度谱。此外，通过反卷积复杂蛋白质丰度谱，我们能够大大提高蛋白质谱的聚类。为了确定假定的相互作用伙伴，并最终确定蛋白质复合物，我们的方法计算蛋白质谱对之间的欧几里得距离。可接受的阈值是基于由接受者-操作者特征（ROC）曲线分析优化的截止值。我们的方法显示出低技术变异，可以很容易地适应于任何生物系统中复杂体的研究。
The majority of cellular processes are carried out by protein complexes. Various size fractionation methods have previously been combined with mass spectrometry to identify protein complexes. However, most of these approaches lack the quantitative information which is required to understand how changes of protein complex abundance and composition affect metabolic fluxes. In this paper we present a proof of concept approach to quantitatively study the complexome in the model plant Arabidopsis thaliana at the end of the day (ED) and the end of the night (EN). We show that size-fractionation of native protein complexes by Clear-Native-PAGE (CN-PAGE), coupled with mass spectrometry can be used to establish abundance profiles along the molecular weight gradient. Furthermore, by deconvoluting complex protein abundance profiles, we were able to drastically improve the clustering of protein profiles. To identify putative interaction partners, and ultimately protein complexes, our approach calculates the Euclidian distance between protein profile pairs. Acceptable threshold values are based on a cut-off that is optimized by a receiver-operator characteristic (ROC) curve analysis. Our approach shows low technical variation and can easily be adapted to study in the complexome in any biological system.