原位生成差异传感器，用于指纹激酶及其表达对细胞反应的影响。
In-situ generation of differential sensors that fingerprint kinases and the cellular response to their expression.
机构信息
Department of Chemistry and Biochemistry and ‡Division of Medicinal Chemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
出版信息
J Am Chem Soc. 2013 Oct 2;135(39):14814-20. doi: 10.1021/ja407397z. Epub 2013 Sep 18.
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.
丝裂原活化蛋白（MAP）激酶负责许多细胞功能，其功能障碍表现在几种人类疾病中。通常，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 Western blot。在此，我们报告了一种用于体外区分 MAP 激酶及其磷酸化对应物的阵列传感方法。该技术利用了原位创建的包含已知与 MAP 激酶具有亲和力的肽的差分受体库，以及与蛋白质上的磷酸基团结合的 Zn(II)-二吡啶基胺络合物。指示剂置换分析信号表明各个受体与激酶的结合，同时化学计量学用于为激酶及其活性状态创建指纹。例如，线性判别分析以 97.5%的体外分类准确率正确识别激酶活性，而对激酶表达的细胞反应则以 100%的准确率进行分类。
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