Peptide microarrays for real-time kinetic profiling of tyrosine phosphatase activity of recombinant phosphatases and phosphatases in lysates of cells or tissue samples

Hovestad-Bijl, L., van Ameijde, J., Pijnenburg, D., Hilhorst, R., Liskamp, R. and Ruijtenbeek, R. (2016) Peptide microarrays for real-time kinetic profiling of tyrosine phosphatase activity of recombinant phosphatases and phosphatases in lysates of cells or tissue samples. In: Pulido, R. (ed.) Protein Tyrosine Phosphatases: Methods and Protocols. Series: Methods in molecular biology, 1447 (1447). Springer New York, pp. 67-78. ISBN 9781493937448 (doi:10.1007/978-1-4939-3746-2_4)

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Abstract

A high-throughput method for the determination of the kinetics of protein tyrosine phosphatase (PTP) activity in a microarray format is presented, allowing real-time monitoring of the dephosphorylation of a 3-nitro-phosphotyrosine residue. The 3-nitro-phosphotyrosine residue is incorporated in potential PTP substrates. The peptide substrates are immobilized onto a porous surface in discrete spots. After dephosphorylation by a PTP, a 3-nitrotyrosine residue is formed that can be detected by a specific, sequence-independent antibody. The rate of dephosphorylation can be measured simultaneously on 12 microarrays, each comprising three concentrations of 48 clinically relevant peptides, using 1.0–5.0 μg of protein from a cell or tissue lysate or 0.1–2.0 μg of purified phosphatase. The data obtained compare well with solution phase assays involving the corresponding unmodified phosphotyrosine substrates. This technology, characterized by high-throughput (12 assays in less than 2 h), multiplexing and low sample requirements, facilitates convenient and unbiased investigation of the enzymatic activity of the PTP enzyme family, for instance by profiling of PTP substrate specificities, evaluation of PTP inhibitors and pinpointing changes in PTP activity in biological samples related to diseases.

Item Type:Book Sections
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Liskamp, Professor Robert
Authors: Hovestad-Bijl, L., van Ameijde, J., Pijnenburg, D., Hilhorst, R., Liskamp, R., and Ruijtenbeek, R.
College/School:College of Science and Engineering > School of Chemistry
Journal Name:Methods in Molecular Biology
Publisher:Springer New York
ISSN:1064-3745
ISSN (Online):1940-6029
ISBN:9781493937448

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