Profiling receptor tyrosine kinase activation by using Ab microarrays
Profiling receptor tyrosine kinase activation by using Ab microarrays
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DOI：
10.1073/pnas.1633513100
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发表时间：
2003-08-05
影响因子：
11.1
通讯作者：
Sorger, PK
中科院分区：
文献类型：
--
作者：
Nielsen, UB;Cardone, MH;Sorger, PK
哺乳动物细胞中的信号转导是由相互作用蛋白质的复杂网络介导的。在电路层面理解这些网络需要设备以快速、准确的方式测量多种蛋白质的数量和活性。 Ab 微阵列先前已应用于标记重组蛋白和血清中蛋白质的定量。微阵列上细胞内信号分子分析方法的发展将使抗体阵列在系统生物学中广泛应用。在这里，我们描述了对粗制细胞裂解物中信号转导蛋白的数量和修饰状态敏感的多重抗体阵列的制造，以及将这些阵列与 96 孔微量滴定板技术集成以在微孔板中创建微阵列。我们应用 Ab 阵列监测人类肿瘤细胞系中 ErbB 受体酪氨酸激酶的激活、摄取和信号传导。从多色比例微阵列获得的数据与使用传统方法获得的数据具有很好的相关性，但阵列速度更快，使用更简单。粗制细胞裂解物的微孔板和微阵列方法的集成应该能够以前所未有的速度和精度识别和分析信号转导过程的小分子抑制剂。我们通过使用 Ab 阵列表征表皮生长因子受体抑制剂 PD153035 对细胞的作用，展示了这种方法的未来潜力；在 96 孔板和 384 孔板中直接放大到基于阵列的筛选应该能够识别出具有针对信号网络的特定抑制谱的小分子。
Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins,and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.