Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics.
Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics.
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DOI：
10.1002/pmic.201400154
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发表时间：
2015-01
期刊：
影响因子：
3.4
通讯作者：
Gygi SP
中科院分区：
文献类型：
--
作者：
Paulo JA;McAllister FE;Everley RA;Beausoleil SA;Banks AS;Gygi SP
基于多重等压标签的定量蛋白质组学和磷酸化蛋白质组学策略可以全面分析药物治疗对生物系统的影响。鉴于MEK信号在癌症和mapk依赖性疾病中的作用，我们试图通过检查下游分子后果来确定是否可以安全地抑制这一途径。我们通过一系列TMT10-plex实验，分析了两种MEK抑制剂（GSK1120212和PD0325901）对9只小鼠三种组织（肾、肝和胰腺）的影响。我们在每个组织中量化了约6000个蛋白质，但在抑制剂治疗下，蛋白质水平的显著变化很小。特别感兴趣的是肾脏组织，因为水肿是这些抑制剂的副作用。从肾脏组织中，我们用二氧化钛（TiO2）富集磷酸肽，并量化了10,562个磷酸化事件。通过磷酸化酪氨酸（pY）肽免疫沉淀进一步分析，量化了另外592个磷酸化事件。磷酸化基序分析显示，抑制剂降低了PxSP和SP位点的磷酸化水平，与ERK抑制一致。MEK抑制剂对参与离子转运和体液平衡的两种蛋白（bartin和Slc12a3）的磷酸化降低幅度最大。进一步的研究将深入了解这些MEK抑制剂在小鼠模型和人类患者中对水肿和其他不良事件的影响。
Multiplexed isobaric tag-based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of MEK signaling in cancer and MAPK-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of TMT10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ~6000 proteins in each tissue, but significant protein level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2) and quantified 10,562 phosphorylation events. Further analysis by phosphotyrosine (pY) peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of PxSP and SP sites, consistent with ERK inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients.