Ramos-Fernández Antonio, López-Ferrer Daniel, Vázquez Jesús
Protein Chemistry and Proteomics Laboratory, Centro de Biología Molecular Severo Ochoa-Consejo Superior de Investigaciones Científicas, 28049 Cantoblanco, Madrid, Spain.
Mol Cell Proteomics. 2007 Jul;6(7):1274-86. doi: 10.1074/mcp.T600029-MCP200. Epub 2007 Feb 23.
Quantitative strategies relying on stable isotope labeling and isotope dilution mass spectrometry have proven to be a very robust alternative to the well established gel-based techniques for the study of the dynamic proteome. Postdigestion 18O labeling is becoming very popular mainly due to the simplicity of the enzyme-catalyzed exchange reaction, the peptide handling and storage procedures, and the flexibility and versatility introduced by decoupling protein digestion from peptide labeling. Despite recent progresses, peptide quantification by postdigestion 18O labeling still involves several computational problems. In this work we analyzed the behavior of large collections of peptides when they were subjected to postdigestion labeling and concluded that this process can be explained by a universal kinetic model. On the basis of this observation, we developed an advanced quantification algorithm for this kind of labeling. Our method fits the entire isotopic envelope to parameters related with the kinetic exchange model, allowing at the same time an accurate calculation of the relative proportion of peptides in the original samples and of the specific labeling efficiency of each one of the peptides. We demonstrated that the new method eliminates artifacts produced by incomplete oxygen exchange in subsets of peptides that have a relatively low labeling efficiency and that may be considered indicative of false protein ratio deviations. Finally using a rigorous statistical analysis based on the calculation of error rates associated with false expression changes, we showed the validity of the method in the practice by detecting significant expression changes, produced by the activation of a model preparation of T cells, with only 5 microg of protein in three proteins among a pool of more than 100. By allowing a full control over potential artifacts, our method may improve automation of the procedures for relative protein quantification using this labeling strategy.
依赖稳定同位素标记和同位素稀释质谱法的定量策略已被证明是研究动态蛋白质组的成熟基于凝胶技术的一种非常可靠的替代方法。消化后18O标记正变得非常流行，主要是由于酶催化交换反应的简单性、肽处理和储存程序，以及通过将蛋白质消化与肽标记解耦所带来的灵活性和通用性。尽管最近取得了进展，但通过消化后18O标记进行肽定量仍涉及几个计算问题。在这项工作中，我们分析了大量肽在进行消化后标记时的行为，并得出该过程可以用一个通用动力学模型来解释的结论。基于这一观察结果，我们为这种标记开发了一种先进的定量算法。我们的方法将整个同位素包络拟合到与动力学交换模型相关的参数上，同时允许准确计算原始样品中肽的相对比例以及每个肽的特定标记效率。我们证明了新方法消除了在标记效率相对较低的肽子集中由于不完全氧交换产生的伪像，这些伪像可能被认为是蛋白质比例偏差错误的指示。最后，通过基于与错误表达变化相关的错误率计算进行严格的统计分析，我们在实践中展示了该方法的有效性，即通过检测仅5微克蛋白质在100多种蛋白质混合物中的三种蛋白质中由T细胞模型制剂激活产生的显著表达变化。通过完全控制潜在的伪像，我们的方法可能会提高使用这种标记策略进行相对蛋白质定量程序的自动化程度。
