Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
J Proteome Res. 2012 Jul 6;11(7):3680-9. doi: 10.1021/pr300147z. Epub 2012 Jun 21.
Label-free methods streamline quantitative proteomics of tissues by alleviating the need for metabolic labeling of proteins with stable isotopes. Here we detail and implement solutions to common problems in label-free data processing geared toward tissue proteomics by one-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectrometry (geLC MS/MS). Our quantification pipeline showed high levels of performance in terms of duplicate reproducibility, linear dynamic range, and number of proteins identified and quantified. When applied to the liver of an adenomatous polyposis coli (APC) knockout mouse, we demonstrated an 8-fold increase in the number of statistically significant changing proteins compared to alternative approaches, including many more previously unidentified hydrophobic proteins. Better proteome coverage and quantification accuracy revealed molecular details of the perturbed energy metabolism.
无标记方法通过减轻用稳定同位素对蛋白质进行代谢标记的需要，简化了组织定量蛋白质组学的工作流程。在这里，我们详细介绍并实施了针对通过一维凝胶电泳后进行液相色谱串联质谱（geLC-MS/MS）的组织蛋白质组学的无标记数据处理中常见问题的解决方案。我们的定量分析流水线在重复重现性、线性动态范围以及鉴定和定量的蛋白质数量方面表现出了很高的性能。当应用于腺瘤性结肠息肉病（APC）基因敲除小鼠的肝脏时，与包括更多先前未鉴定的疏水性蛋白质在内的替代方法相比，我们证明了统计学上显著变化的蛋白质数量增加了 8 倍。更好的蛋白质组覆盖范围和定量准确性揭示了受干扰的能量代谢的分子细节。
