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

Authors

  • Dr. Jeroen van Ameijde,

    1. Medicinal Chemistry and Chemical Biology, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht (The Netherlands), Fax: (+31) (0)30-253-6655
    2. Netherlands Proteomics Centre, Padualaan 8, 3584 CA Utrecht (The Netherlands)
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  • Dr. John Overvoorde,

    1. Hubrecht Institute, KNAW and University Medical Centre, Uppsalalaan 8, 3508 AD Utrecht (The Netherlands)
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  • Prof. Dr. Stefan Knapp,

    1. Structural Genomics Consortium, Oxford University, Roosevelt Drive, Headington, Oxford OX3 7DQ (U.K.)
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  • Prof. Dr. Jeroen den Hertog,

    1. Hubrecht Institute, KNAW and University Medical Centre, Uppsalalaan 8, 3508 AD Utrecht (The Netherlands)
    2. Institute of Biology, Leiden University, P.O. Box 9502, 2300 RA Leiden (The Netherlands)
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  • Dr. Rob Ruijtenbeek,

    1. Pamgene International Ltd. Wolvenhoek 10, 5200 BJ Den Bosch (The Netherlands)
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  • Prof. Dr. Rob M. J. Liskamp

    Corresponding author
    1. Medicinal Chemistry and Chemical Biology, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht (The Netherlands), Fax: (+31) (0)30-253-6655
    2. School of Chemistry, Joseph Black Building, Glasgow University, University Avenue, Glasgow G12 8QQ (U.K.)
    • Medicinal Chemistry and Chemical Biology, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht (The Netherlands), Fax: (+31) (0)30-253-6655

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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.

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