Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients
Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients
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DOI：
10.1038/nprot.2012.036
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发表时间：
2012-04-01
期刊：
影响因子：
14.8
通讯作者：
Mechtler, Karl
中科院分区：
文献类型：
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作者：
Koecher, Thomas;Pichler, Peter;Mechtler, Karl
大多数蛋白质组研究依赖于高分离能力的二维液相色谱-串联质谱（2D LCLC-MS/MS），通常与蛋白质预分离相结合。为了减少分析时间和所需的样品量，替代方法将是有利的。在色谱和质谱仪器的最新进展的基础上，成千上万的蛋白质可以确定在一个单一的运行LCLC-MS/MS实验使用超长梯度。因此，简单的蛋白质组或临床样本的分析在足够的深度成为可能，通过执行单次运行LCLC-MS/MS实验。在这里，我们提出了一个普遍适用的协议，从未分离的全细胞提取物的蛋白质分析，并讨论其潜力和局限性。为了证明该方法的实用性，我们从HeLa细胞裂解物中鉴定了2，761种蛋白质，需要大约10小时的nanoLCLC-MS/MS测量时间。
The majority of proteome-wide studies rely on the high separation power of two-dimensional liquid chromatography-tandem mass spectrometry (2D LCLC-MS/MS), often combined with protein prefractionation. Alternative approaches would be advantageous in order to reduce the analysis time and the amount of sample required. On the basis of the recent advances in chromatographic and mass spectrometric instrumentation, thousands of proteins can be identified in a single-run LCLC-MS/MS experiment using ultralong gradients. Consequently, the analysis of simple proteomes or clinical samples in adequate depth becomes possible by performing single-run LCLC-MS/MS experiments. Here we present a generally applicable protocol for protein analysis from unseparated whole-cell extracts and discuss its potential and limitations. Demonstrating the practical applicability of the method, we identified 2,761 proteins from a HeLa cell lysate, requiring around 10 h of nanoLCLC-MS/MS measurement time.