R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies
R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies
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DOI：
10.15252/msb.20199021
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发表时间：
2019-12-01
影响因子：
9.9
通讯作者：
Villen, Judit
中科院分区：
文献类型：
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作者：
Leutert, Mario;Rodriguez-Mias, Ricard A.;Villen, Judit
蛋白质组学的最新发展使信号传导研究成为可能，其中可以常规地鉴定和定量> 10，000个磷酸化位点。然而，目前的分析在通量、再现性和鲁棒性方面受到限制，妨碍了涉及多种扰动的实验，例如映射激酶-底物关系、捕获途径串扰和网络推理分析所需的那些。为了应对这些挑战，我们引入了快速机器人磷酸蛋白质组学（R2-P2），这是一种端到端的自动化方法，使用磁性颗粒处理蛋白质提取物，以提供质谱分析准备的磷酸肽。R2-P2具有快速、稳健、多功能和高通量的特点。为了展示该方法，我们将其与数据独立采集质谱法相结合，用于研究酵母中丝裂原活化蛋白激酶（MAPK）途径的信号动力学。我们的研究结果揭示了广泛的和特定的信号事件沿着交配，高渗透压甘油，和侵袭性生长分支的MAPK途径，与强大的磷酸化下游调控蛋白和转录因子。我们的方法有助于涉及数百个扰动的大规模信号研究，为旨在捕获信号复杂性的系统级研究打开了大门。
Recent developments in proteomics have enabled signaling studies where > 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid-robotic phosphoproteomics (R2-P2), an end-to-end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry-ready phosphopeptides. R2-P2 is rapid, robust, versatile, and high-throughput. To showcase the method, we applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large-scale signaling studies involving hundreds of perturbations opening the door to systems-level studies aiming to capture signaling complexity.