Mass spectrometry has transformed the field of cell signaling by enabling global studies of dynamic protein phosphorylation ('phosphoproteomics'). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these and is sufficiently streamlined to enable the analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated workflow that further ensures high performance in sample-limited conditions while also reducing sample preparation time. By eliminating protein precipitation steps and performing the entire protocol, including digestion, in a single 96-well plate, we now greatly minimize opportunities for sample loss and variability. This results in very high reproducibility and a small sample size requirement (≤200 μg of protein starting material). After cell culture or tissue collection, the protocol takes 1 d, whereas mass spectrometry measurements require ~1 h per sample. Applied to glioblastoma cells acutely treated with epidermal growth factor (EGF), EasyPhos quantified 20,132 distinct phosphopeptides from 200 μg of protein in less than 1 d of measurement time, revealing thousands of EGF-regulated phosphorylation events.

中文翻译：

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使用 EasyPhos 平台的高通量和高灵敏度磷酸化蛋白质组学。

质谱法通过实现动态蛋白质磷酸化（“磷酸蛋白质组学”）的全球研究，改变了细胞信号传导领域。最近的发展使得越来越复杂的磷酸化蛋白质组学研究成为可能，但实际挑战仍然存在。EasyPhos 工作流程解决了这些问题，并且经过充分简化，可以在超过 10,000 个量化磷酸化位点的深度分析数百个磷酸化蛋白质组。在这里，我们展示了一个详细且更新的工作流程，可进一步确保在样品有限条件下的高性能，同时减少样品制备时间。通过消除蛋白质沉淀步骤并在单个 96 孔板中执行包括消化在内的整个协议，我们现在极大地减少了样品丢失和变异的机会。这导致非常高的重现性和小样本量要求（≤200 μg 蛋白质起始材料）。在细胞培养或组织收集后，该协议需要 1 天，而质谱测量需要每个样本约 1 小时。应用于经表皮生长因子 (EGF) 急性处理的胶质母细胞瘤细胞，EasyPhos 在不到 1 天的测量时间内从 200 μg 蛋白质中量化了 20,132 个不同的磷酸肽，揭示了数千个 EGF 调节的磷酸化事件。