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