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

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.

Array‐based “Chemical Nose” Sensing in Diagnostics and Drug Discovery

“…All rights reserved. Anslyn et al monitored the post-translational phosphorylation and subsequent activation of kinases in cell lysates as a tool for studying the kinase signaling and apoptosis pathways 67 . As with the work of Waters, Anslyn created a dynamic supramolecular array with three components: a hydrogen-bonding molecule, a Zn 2+ complex mounted in various configurations on a three-armed supramolecular receptor complex and two cross-reactive peptide recognition sequences for their target.…”

Achieving High Affinity and Selectivity for Asymmetric Dimethylarginine by Putting a Lid on a Box

“…These PTMs are of significant interest due to their role in regulating gene expression and their dysregulation in numerous diseases . Much work has gone into developing synthetic receptors that bind PTMs selectively in aqueous solution, with applications in sensing, tagging, enrichment, and enzymatic assays . However, achieving selectivity for one methylation state of Lys or Arg has been a persisting challenge, since all eight possible methylation states can participate in electrostatic interactions, cation–π interactions, and H‐bonding, with the exception of trimethyllysine (Kme3) which cannot H‐bond.…”

Array‐basierte Sensorik mit der “chemischen Nase” in der Diagnostik und Wirkstoffentdeckung

“…Anslyn et al. verfolgten die posttranslationale Phosphorylierung und nachfolgende Aktivierung von Kinasen in Zelllysaten, um Kinasesignalgebung und Apoptosepfade zu studieren . Wie Waters et al.…”