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.

中文翻译：

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R2-P2 快速机器人磷酸蛋白质组学可实现多维细胞信号传导研究。

蛋白质组学的最新发展使得信号研究成为可能，其中可以常规鉴定和定量超过 10,000 个磷酸位点。然而，当前的分析在通量、重现性和鲁棒性方面受到限制，阻碍了涉及多种扰动的实验，例如绘制激酶-底物关系、捕获通路串扰和网络推理分析所需的实验。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)，这是一种端到端自动化方法，使用磁性颗粒处理蛋白质提取物，以提供可供质谱分析的磷酸肽。R2-P2 快速、稳健、多功能且高通量。为了展示该方法，我们将其与数据无关的采集质谱相结合，研究酵母中丝裂原激活蛋白激酶 (MAPK) 途径的信号动力学。我们的结果揭示了沿着 MAPK 途径的交配、高渗透甘油和侵入生长分支的广泛而特异的信号传导事件，以及下游调节蛋白和转录因子的强磷酸化。我们的方法促进了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。