Real-Time Monitoring of the Dephosphorylating Activity of Protein Tyrosine Phosphatases Using Microarrays with 3-Nitrophosphotyrosine Substrates

Phosphatases and kinases regulate the crucial phosphorylation post-translational modification. In spite of their similarly important role in many diseases and therapeutic potential, phosphatases have received arguably less attention. One reason for this is a scarcity of high-throughput phosphatase assays. Herein, a new real-time, dynamic protein tyrosine phosphatase (PTP) substrate microarray assay measuring product formation is described. PTP substrates comprising a novel 3-nitrophosphotyrosine residue are immobilized in discrete spots. After reaction catalyzed by a PTP a 3-nitrotyrosine residue is formed that can be detected by specific, sequence-independent antibodies. The resulting microarray was successfully evaluated with a panel of recombinant PTPs and cell lysates, which afforded results comparable to data from other assays. Its parallel nature, convenience, and low sample requirements facilitate investigation of the therapeutically relevant PTP enzyme family. Keeping it real: The activity of important protein tyrosine phosphatases has been monitored in real time in parallel with a novel substrate microarray through formation of 3-nitrotyrosine (see figure). Copyright © 2013 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim.