Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells
Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells
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DOI：
10.1038/nmeth.2834
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发表时间：
2014-03-01
期刊：
影响因子：
48
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Kulak, Nils A.;Pichler, Garwin;Mann, Matthias
基于质谱（MSMS）的蛋白质组学通常采用多步样品制备工作流程，其易受样品污染和损失。我们报告了一种用于进行样品处理的In-StageTip方法，从细胞裂解到纯化肽的洗脱，在一个单一的封闭体积中。这种强大且可扩展的方法在很大程度上消除了污染或损失。肽可以在几个馏分中洗脱或在一个步骤中进行单次运行蛋白质组分析。在一天之内，我们获得了最大的蛋白质组覆盖率到目前为止的芽殖和裂殖酵母，并发现这些细胞中的蛋白质拷贝数是高度相关的（R-2 = 0.78）。应用in-StageTip方法对人类细胞系进行四倍体测量，我们获得了9，667种人类蛋白质的拷贝数估计值，并观察到重复之间具有良好的定量重现性（R-2 = 0.97）。In-StageTip方法简单明了，通常适用于生物学或临床应用。
Mass spectrometry (MSMS)-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.