Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli
Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli
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DOI：
10.1016/j.jprot.2012.06.020
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发表时间：
2012-09-18
影响因子：
3.3
通讯作者：
Vilu, R.
中科院分区：
文献类型：
--
作者：
Arike, L.;Valgepea, K.;Vilu, R.
评估了三种不同的无标记蛋白质组定量方法-APEX，emPAI和iBAQ-以测量细胞中蛋白质组范围的蛋白质浓度。所有的方法都适用于从大肠杆菌恒化培养的样品。证明了三种定量方法之间的皮尔逊平方相关性约为0.6。重要的是，通过iBAQ和emPAI定量的蛋白质的总和与Lowry总蛋白质定量相对应，证明了无标记方法在蛋白质组水平准确计算蛋白质浓度的适用性。iBAQ方法显示了生物重复之间的最佳相关性，所有蛋白质丰度之间的正态分布，以及核糖体蛋白质丰度之间的最低变化，预期具有相等amounts.绝对定量蛋白质组数据使我们能够通过与通量分析的整合来评估蛋白质合成的代谢成本和酶的表观催化活性。所有的方法都证明了不同细胞过程的蛋白质合成的ATP成本相似，并且表达生物质合成相关蛋白质的成本高于能量产生的成本。重要的是，能量代谢酶的催化活性比单体合成酶高一两个数量级。有趣的是，阶梯状蛋白质表达被证明为大多数的转录单位。(C)2012爱思唯尔有限公司版权所有。
Three different label-free proteome quantification methods - APEX, emPAI and iBAQ - were evaluated to measure proteome-wide protein concentrations in the cell. All the methods were applied to a sample from Escherichia coli chemostat culture. A Pearson squared correlation of approximately 0.6 among the three quantification methods was demonstrated. Importantly, the sum of quantified proteins by iBAQ and emPAI corresponded with the Lowry total protein quantification, demonstrating applicability of label-free methods for an accurate calculation of protein concentrations at the proteome level. The iBAQ method showed the best correlation between biological replicates, a normal distribution among all protein abundances, and the lowest variation among ribosomal protein abundances, which are expected to have equal amounts.Absolute quantitative proteome data enabled us to evaluate metabolic cost for protein synthesis and apparent catalytic activities of enzymes by integration with flux analysis. All the methods demonstrated similar ATP costs for protein synthesis for different cellular processes and that costs for expressing biomass synthesis related proteins were higher than those for energy generation. Importantly, catalytic activities of energy metabolism enzymes were an order or two higher than those of monomer synthesis. Interestingly, a staircase-like protein expression was demonstrated for most of the transcription units. (C) 2012 Elsevier B.V. All rights reserved.