Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes
Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes
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DOI：
10.1021/pr050406g
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发表时间：
2006-05-01
影响因子：
4.4
通讯作者：
Shen, RF
中科院分区：
文献类型：
--
作者：
Wang, GH;Wu, WW;Shen, RF
蛋白质生物标志物发现的关键步骤是对比蛋白质组的能力，这一过程通常被称为定量蛋白质组学。虽然稳定同位素标记（例如，ICAT，O-18-或N-15-标记，或AQUA）仍然是基于质谱的蛋白质组学定量中使用的核心技术，越来越多的努力已经指向依赖于直接比较LC-MS运行之间的肽峰面积的无标记方法。后一种方法因其简单性和成本效益而对调查人员具有吸引力。在本研究中，使用无标记的方法来高度复杂的蛋白质组的重复性和线性进行了评估。使用离子阱或傅里叶变换质谱仪对来自不同蛋白质组的不同量的蛋白质进行重复LC-MS分析。在重复运行之间获得了高度可重复的数据，如由接近理想的Pearson相关系数（对于离子的峰面积或保留时间）和平均峰面积比所证明的。一般而言，超过50%和近90%的肽离子比率分别偏离两次运行的平均值小于10%和20%。此外，所用蛋白质量的多重比与从检测到的肽计算的峰面积的观察到的平均比很好地相关。此外，从样品中去除丰富的蛋白质导致重现性和线性的改善。已编写了一个计算机程序，用于自动处理来自多个样品组的实验数据集，以进行统计分析。采用抗离群值均值估计和调整多重检验统计学显著性阈值的算法，以最大限度地降低假阳性率。该程序被应用于定量蛋白质组的父母和p53缺陷的HCT-116人类细胞的变化，并发现产生可重复的结果。总体而言，这项研究证明了一种替代方法，允许在复杂的蛋白质组差异表达的蛋白质的全球定量。该方法在生物标志物发现中的实用性可能与质谱仪检测灵敏度的未来改进协同作用。
A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, O-18- or N-15-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.