Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
Nat Methods. 2014 Mar;11(3):319-24. doi: 10.1038/nmeth.2834. Epub 2014 Feb 2.
Mass spectrometry (MS)-based proteomics typically employs multistep sample-preparation workflows that are subject to sample contamination and loss. We report an in-StageTip method for performing sample processing, from cell lysis through elution of purified peptides, in a single, enclosed volume. This robust and scalable method largely eliminates contamination or loss. Peptides can be eluted in several fractions or in one step for single-run proteome analysis. In one day, we obtained the largest proteome coverage to date for budding and fission yeast, and found that protein copy numbers in these cells were highly correlated (R(2) = 0.78). Applying the in-StageTip method to quadruplicate measurements of a human cell line, we obtained copy-number estimates for 9,667 human proteins and observed excellent quantitative reproducibility between replicates (R(2) = 0.97). The in-StageTip method is straightforward and generally applicable in biological or clinical applications.
基于质谱（MS）的蛋白质组学通常采用多步样品制备工作流程，这些流程容易受到样品污染和损失的影响。我们报告了一种在 StageTip 上进行样品处理的方法，从细胞裂解到纯化肽的洗脱，都在一个封闭的单一体积中进行。这种稳健且可扩展的方法在很大程度上消除了污染或损失。肽可以分几个馏分洗脱，也可以一步洗脱进行单次运行的蛋白质组分析。在一天内，我们获得了酿酒酵母和裂殖酵母迄今为止最大的蛋白质组覆盖率，并发现这些细胞中的蛋白质拷贝数高度相关（R² = 0.78）。将 in-StageTip 方法应用于人类细胞系的四重测量，我们获得了 9667 个人类蛋白质的拷贝数估计值，并观察到重复之间具有出色的定量重现性（R² = 0.97）。in-StageTip 方法简单直接，在生物学或临床应用中具有广泛的适用性。
