López-Ferrer Daniel, Ramos-Fernández Antonio, Martínez-Bartolomé Salvador, García-Ruiz Predestinación, Vázquez Jesús
Protein Chemistry and Proteomics Laboratory, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
Proteomics. 2006 Apr;6 Suppl 1:S4-11. doi: 10.1002/pmic.200500375.
Quantitative proteomics using stable isotopic 16O/18O labeling has emerged as a very powerful tool, since it has a number of advantages over other methods, including the simplicity of chemistry, the constant mass tag at the C termini and its general applicability. However, due to the small mass difference between labeled and unlabeled peptide species, this approach has usually been restricted to high-resolution mass spectrometers. In this study we explored whether the high-resolution scanning mode, together with the extremely high scanning speed of the linear IT allows the 16O/18O-labeling method to be used for accurate, large-scale quantitative analysis of proteomes. A protocol, including digestion, desalting, labeling, MS and quantitative analysis was developed and tested using protein standards and whole proteome extracts. Using this method we were able to identify and quantify 140 proteins from only 10 mug of a proteome extract from mesenchymal stem cells. Relative expression changes larger than twofold can be identified with this method at the 95% confidence level. Our results demonstrate that accurate quantitative analysis using 16O/18O labeling can be performed in the practice using linear IT MS, without compromising large-scale peptide identification efficiency.
使用稳定同位素16O/18O标记的定量蛋白质组学已成为一种非常强大的工具，因为它相对于其他方法具有许多优势，包括化学操作简单、C端恒定质量标签及其普遍适用性。然而，由于标记和未标记肽段之间的质量差异较小，这种方法通常仅限于高分辨率质谱仪。在本研究中，我们探讨了高分辨率扫描模式以及线性离子阱极高的扫描速度是否能使16O/18O标记方法用于蛋白质组的准确、大规模定量分析。我们开发了一种包括消化、脱盐、标记、质谱和定量分析的方案，并使用蛋白质标准品和全蛋白质组提取物进行了测试。使用这种方法，我们能够从仅10微克间充质干细胞蛋白质组提取物中鉴定和定量140种蛋白质。在95%置信水平下，使用该方法可以鉴定出大于两倍的相对表达变化。我们的结果表明，使用线性离子阱质谱在实际操作中可以进行基于16O/18O标记的准确定量分析，而不会影响大规模肽段鉴定效率。
