In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.
In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.
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DOI：
10.1021/ja407397z
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发表时间：
2013-10-02
影响因子：
15
通讯作者：
Anslyn EV
中科院分区：
文献类型：
--
作者：
Zamora-Olivares D;Kaoud TS;Dalby KN;Anslyn EV
丝裂原活化蛋白(MAP)激酶负责许多细胞功能，它们的故障在几种人类疾病中表现出来。通常，在体外监测MAP激酶的磷酸化状态需要蛋白质的制备和纯化或蛋白质印迹。在这里，我们报告了一种阵列传感方法，用于在体外区分MAP激酶和它们的磷酸化对应物。这项技术利用了一个原位创建的差异受体文库，其中包含以亲和力著称的多肽来映射激酶，以及一个锌(II)-二吡啶甲胺复合体，它与蛋白质上的磷酸基团结合。指示剂置换试验发出信号，表明个别受体与激酶结合，而化学计量学则用于为激酶及其活性状态创建指纹。例如，线性判别分析在体外正确识别了激酶活性，分类准确率为97.5%，而细胞对激酶表达的反应分类准确率为100%。
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)–dipicolylamine 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.
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