Protein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward. Herein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. This platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy.

中文翻译：

---

使用nanoLC-MS/MS和DIA方法开发高通量激酶活性平台，用于研究紫杉醇在卵巢癌中的抗癌机制


蛋白激酶的蛋白质磷酸化在增加蛋白质多样性方面发挥着关键作用，从而影响各种细胞功能。然而，由于肽混合物中磷酸肽的丰度相对较低以及非磷酸化肽的离子抑制效应，磷酸肽的检测并不简单。在此，开发了一个定量高通量平台，用于使用纳米 LC-MS/MS 和数据独立采集 (DIA) 方法评估多激酶活性。通过研究紫杉醇处理的 SKOV3 细胞中的激酶活性来评估该平台。设计并分析了包含 38 种肽底物的文库，以确定参与癌症发展的主要激酶的活性。二十三种合成肽底物在三次生物实验中显示出显着的磷酸化变化，并通过蛋白质印迹进一步验证。我们的研究结果表明，紫杉醇通过激活 AMPK 并抑制 PI3K-Akt 依赖性途径来抑制 SKOV3 细胞存活，最终导致 mTOR 抑制。此外，紫杉醇与ERK、Akt、SGK、CK1和ErbB2抑制剂联合使用，增强了对卵巢癌的抑制作用。该平台对于大规模激酶活性研究来说是一种有吸引力的方法，可以全面揭示药物疾病治疗的机制并研究更有效的治疗策略。