Single-step Enrichment by Ti4+-IMAC and Label-free Quantitation Enables In-depth Monitoring of Phosphorylation Dynamics with High Reproducibility and Temporal Resolution
Single-step Enrichment by Ti4+-IMAC and Label-free Quantitation Enables In-depth Monitoring of Phosphorylation Dynamics with High Reproducibility and Temporal Resolution
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DOI：
10.1074/mcp.o113.036608
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发表时间：
2014-09-01
影响因子：
7
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
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作者：
de Graaf, Erik L.;Giansanti, Piero;Heck, Albert J. R.
定量磷酸化蛋白质组学工作流程传统上涉及额外的样品标记和分馏步骤，以进行准确和深入的分析。在这里，我们报告了一个高通量，简单，全面的无标记磷酸化蛋白质组学方法，使用高选择性，可重复性和敏感的钛4 +-IMAC磷酸肽富集方法。我们通过监测前列腺素E-2（PGE（2））刺激的Jurkat T细胞在6个不同时间点的磷酸化蛋白质组动力学，总共测量108个磷酸化蛋白质组快照，证明了这种方法的适用性。总的来说，我们在所有时间点定量监测了12，799个独特的磷酸位点，具有非常高的定量再现性（100次测量的平均r > 0.9，中值CV < 0.2）。已知PGE（2）可增加细胞cAMP水平，从而激活PKA。深入的分析揭示了各种各样的磷酸化位点的时间调节，不仅与PKA，而且与各种其他类别的激酶。在PGE（2）刺激后，几条通路仅被短暂激活，这表明深入的动态分析需要具有高时间分辨率的技术。此外，大量公开可用的数据集为T细胞中下游PGE（2）信号传导动力学，特别是cAMP介导的信号传导提供了宝贵的资源。更一般地说，我们的方法能够在任何系统上进行深入，定量，高通量的磷酸化蛋白质组筛选，需要非常少的样品，样品制备和分析时间。
Quantitative phosphoproteomics workflows traditionally involve additional sample labeling and fractionation steps for accurate and in-depth analysis. Here we report a high-throughput, straightforward, and comprehensive label-free phosphoproteomics approach using the highly selective, reproducible, and sensitive Ti4+-IMAC phosphopeptide enrichment method. We demonstrate the applicability of this approach by monitoring the phosphoproteome dynamics of Jurkat T cells stimulated by prostaglandin E-2 (PGE(2)) over six different time points, measuring in total 108 snapshots of the phosphoproteome. In total, we quantitatively monitored 12,799 unique phosphosites over all time points with very high quantitative reproducibility (average r > 0.9 over 100 measurements and a median cv < 0.2). PGE(2) is known to increase cellular cAMP levels, thereby activating PKA. The in-depth analysis revealed temporal regulation of a wide variety of phosphosites associated not only with PKA, but also with a variety of other classes of kinases. Following PGE(2) stimulation, several pathways became only transiently activated, revealing that in-depth dynamic profiling requires techniques with high temporal resolution. Moreover, the large publicly available dataset provides a valuable resource for downstream PGE(2) signaling dynamics in T cells, and cAMP-mediated signaling in particular. More generally, our method enables in-depth, quantitative, high-throughput phosphoproteome screening on any system, requiring very little sample, sample preparation, and analysis time.