Real-time monitoring of the dephosphorylating activity of protein tyrosine phosphatases using microarrays with 3-nitrophosphotyrosine substrates

van Ameijde, J., Overvoorde, J., Knapp, S., den Hertog, J., Ruijtenbeek, R. and Liskamp, R. M. J. (2013) Real-time monitoring of the dephosphorylating activity of protein tyrosine phosphatases using microarrays with 3-nitrophosphotyrosine substrates. ChemPlusChem, 78(11), pp. 1349-1357. (doi: 10.1002/cplu.201300299)

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Liskamp, Professor Robert
• Authors: van Ameijde, J., Overvoorde, J., Knapp, S., den Hertog, J., Ruijtenbeek, R., and Liskamp, R. M. J.
• College/School: College of Science and Engineering > School of Chemistry
• Journal Name: ChemPlusChem
• Publisher: Wiley-VCH Verlag
• ISSN: 2192-6506
• ISSN (Online): 2192-6506