Phosphorylation is a post-translational modification in proteins that changes protein conformation and activity for regulating signal transduction pathways. This mechanism is frequently impaired in lung cancer, resulting in permanently active constitutive phosphorylation to initiate tumor growth and/or reactivate pathways in response to therapy. We developed a multiplexed phosphoprotein analyzer chip (MPAC) that enables rapid (detection time: 5 min) and sensitive (LOD: 2 pg/μL) detection of protein phosphorylation and presents phosphoproteomic profiling of major phosphorylation pathways in lung cancer. We monitored phosphorylated receptors and downstream proteins involved in mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR pathways in lung cancer cell line models and patient-derived extracellular vesicles (EV). Using kinase inhibitor drugs in cell line models, we found that the drug can inhibit the phosphorylation and/or activation of the kinase pathway. We then generated a phosphorylation heatmap by EV phosphoproteomic profiling of plasma samples isolated from 36 lung cancer patients and 8 noncancer individuals. The heatmap showed a clear difference between the noncancer and cancer samples and identify the specific proteins that are activated in the cancer samples. Our data also showed that MPAC could monitor immunotherapy responses by assessment of the phosphorylation states of the proteins, particularly for PD-L1. Finally, with a longitudinal study, we found that the phosphorylation levels of the proteins were indicative of a positive response to therapy. We believe that this study will lead to personalized treatment by providing a better understanding of the active and resistant pathways and will provide a tool for selecting combined and targeted therapies for precision medicine.

中文翻译：

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用于诊断和治疗监测的肺癌患者关键磷酸化蛋白质组学途径的液体活检快照

磷酸化是蛋白质的翻译后修饰，它改变蛋白质的构象和活性以调节信号转导途径。这种机制在肺癌中经常受损，导致永久活跃的组成型磷酸化以启动肿瘤生长和/或重新激活通路以响应治疗。我们开发了一种多路复用磷蛋白分析芯片 (MPAC)，可以快速（检测时间：5 分钟）和灵敏（LOD：2 pg/μL）检测蛋白质磷酸化，并呈现肺癌主要磷酸化途径的磷酸化蛋白质组学分析。我们监测了肺癌细胞系模型和患者来源的细胞外囊泡 (EV) 中参与丝裂原活化蛋白激酶 (MAPK) 和 PI3K/AKT/mTOR 通路的磷酸化受体和下游蛋白。在细胞系模型中使用激酶抑制剂药物，我们发现该药物可以抑制激酶通路的磷酸化和/或激活。然后，我们通过对从 36 名肺癌患者和 8 名非癌症个体中分离出的血浆样本进行 EV 磷酸化蛋白质组学分析，生成了磷酸化热图。热图显示了非癌症和癌症样本之间的明显差异，并确定了在癌症样本中被激活的特定蛋白质。我们的数据还表明，MPAC 可以通过评估蛋白质（尤其是 PD-L1）的磷酸化状态来监测免疫治疗反应。最后，通过纵向研究，我们发现蛋白质的磷酸化水平表明对治疗有积极反应。