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Real‐Time Monitoring of the Dephosphorylating Activity of Protein Tyrosine Phosphatases Using Microarrays with 3‐Nitrophosphotyrosine Substrates
Author(s) -
van Ameijde Jeroen,
Overvoorde John,
Knapp Stefan,
den Hertog Jeroen,
Ruijtenbeek Rob,
Liskamp Rob M. J.
Publication year - 2013
Publication title -
chempluschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.801
H-Index - 61
ISSN - 2192-6506
DOI - 10.1002/cplu.201300299
Subject(s) - protein tyrosine phosphatase , phosphatase , phosphorylation , chemistry , biochemistry , kinase , dna microarray , tyrosine , enzyme , proteomics , computational biology , biology , gene , gene expression
Abstract 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.