In-Situ Generation of Differential Sensors that Fingerprint Kinases and the Cellular Response to Their Expression

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• 作者：Diana Zamora-Olivares ; Tamer S. Kaoud ; Kevin N. Dalby ; Eric V. Anslyn
• 刊名：Journal of the American Chemical Society
• 出版年：2013
• 出版时间：October 2, 2013
• 年：2013
• 卷：135
• 期：39
• 页码：14814-14820
• 全文大小：423K
• 年卷期：v.135,no.39(October 2, 2013)
• ISSN：1520-5126

文摘

Mitogen-activated protein (MAP) kinases are responsible for many cellular functions, and their malfunction manifests itself in several human diseases. Usually, monitoring the phosphorylation states of MAP kinases in vitro requires the preparation and purification of the proteins or Western blotting. Herein, we report an array sensing approach for the differentiation of MAP kinases and their phosphorylated counterparts in vitro. This technique utilizes a library of differential receptors created in situ containing peptides known for affinity to MAP kinases, and a Zn(II)鈥揹ipicolylamine complex that binds phosphate groups on proteins. An indicator-displacement assay signals the binding of the individual receptors to the kinases, while chemometrics is used to create a fingerprint for the kinases and their state of activity. For example, linear discriminant analysis correctly identified kinase activity with a classification accuracy of 97.5% in vitro, while the cellular response to kinase expression was classified with 100% accuracy.