Proteomic analysis of rat liver phosphoproteins after treatment with protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide)
Proteomic analysis of rat liver phosphoproteins after treatment with protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide)
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DOI：
10.1124/jpet.105.100032
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发表时间：
2006-08-01
影响因子：
3.5
通讯作者：
Tam, Sun W.
中科院分区：
文献类型：
--
作者：
Davis, Myrtle A.;Hinerfeld, Douglas;Tam, Sun W.
专注于激酶抑制的治疗策略在很大程度上依赖于从体外测定系统获得的激酶抑制的替代措施。有必要开发一种方法，以方便测量用这些化合物处理过的整个动物组织样品中的激酶抑制剂活性或特异性。目前的许多方法都受到抗体使用的限制，其中许多抗体不能在几个物种之间交叉反应。本文描述的蛋白质组学方法有可能通过监测蛋白质磷酸化的差异来揭示新的组织底物、潜在的新途径相互联系和抑制剂特异性。我们使用蛋白激酶抑制剂H89 （N-（2-[对溴肉桂胺]乙基）-5-异喹啉磺酰胺）作为工具来确定组织磷酸化蛋白的差异谱是否可以用于检测蛋白激酶a （PKA）抑制剂在体内的治疗相关作用。通过磷蛋白柱富集、高通量二维凝胶电泳、Pro-Q Diamond/SYPRO Ruby差异凝胶染色、统计分析和基质辅助激光解吸电离/飞行时间质谱分析，我们能够显示对照组和处理过的大鼠肝蛋白提取物的磷蛋白谱存在明显差异。此外，一些显示出潜在磷酸化变化的蛋白先前被鉴定为PKA底物或具有假定的PKA磷酸化位点。提出的数据支持使用差异蛋白质组学方法来测量激酶抑制剂治疗对体内蛋白质磷酸化的影响。
Therapeutic strategies focused on kinase inhibition rely heavily on surrogate measures of kinase inhibition obtained from in vitro assay systems. There is a need to develop methodology that will facilitate measurement of kinase inhibitor activity or specificity in tissue samples from whole animals treated with these compounds. Many of the current methods are limited by the use of antibodies, many of which do not cross-react between several species. The proteomics approach described herein has the potential to reveal novel tissue substrates, potential new pathway interconnections, and inhibitor specificity by monitoring differences in protein phosphorylation. We used the protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide) as a tool to determine whether differential profiling of tissue phosphoproteins can be used to detect treatment-related effects of a protein kinase A (PKA) inhibitor in vivo. With a combination of phosphoprotein column enrichment, high-throughput two-dimensional gel electrophoresis, differential gel staining with Pro-Q Diamond/SYPRO Ruby, statistical analysis, and matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis, we were able to show clear differences between the phosphoprotein profiles of rat liver protein extract from control and treated animals. Moreover, several proteins that show a potential change in phosphorylation were previously identified as PKA substrates or have putative PKA phosphorylation sites. The data presented support the use of differential proteomic methods to measure effects of kinase inhibitor treatment on protein phosphorylation in vivo.