title: Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples.
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.
authors: PamGene International BV, 1345, 5200 BJ, 's-Hertogenbosch, The Netherlands.
authors: Liesbeth
authors: L
authors: Hovestad-Bijl
authors: Janssen Prevention Center, Crucell Holland BV, Leiden, The Netherlands.
authors: Jeroen
authors: J
authors: van Ameijde
authors: PamGene International BV, 1345, 5200 BJ, 's-Hertogenbosch, The Netherlands.
authors: Dirk
authors: D
authors: Pijnenburg
authors: PamGene International BV, 1345, 5200 BJ, 's-Hertogenbosch, The Netherlands.
authors: Riet
authors: R
authors: Hilhorst
authors: School of Chemistry, University of Glasgow, Glasgow, UK.|Medicinal Chemistry and Chemical Biology, Utrecht University, Utrecht, The Netherlands.
authors: Rob
authors: R
authors: Liskamp
authors: PamGene International BV, 1345, 5200 BJ, 's-Hertogenbosch, The Netherlands. rruijtenbeek@pamgene.com.
authors: Rob
authors: R
authors: Ruijtenbeek
mesh_terms: D000818:Animals; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases
keywords: Multiplex assay; Nitrotyrosine; Peptide microarray; Phosphatase activity; Phosphatase activity profiling; Phosphatase inhibition; Phosphatase kinetics; Phosphatase substrate identification; Phospho-nitrotyrosine; Tyrosine phosphatase