Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples

  • Liesbeth Hovestad-Bijl
  • Jeroen van Ameijde
  • Dirk Pijnenburg
  • Riet Hilhorst
  • Rob Liskamp
  • Rob Ruijtenbeek
Part of the Methods in Molecular Biology book series (MIMB, volume 1447)


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.

Key words

Tyrosine phosphatase Phosphatase activity Peptide microarray Multiplex assay Phosphatase substrate identification Phosphatase activity profiling Phosphatase kinetics Phosphatase inhibition Phospho-nitrotyrosine Nitrotyrosine 


  1. 1.
    Hatzihristidis T, Liu S, Pryszcz L et al (2014) PTP-central: a comprehensive resource of protein tyrosine phosphatases in eukaryotic genomes. Methods 65:156–164CrossRefPubMedGoogle Scholar
  2. 2.
    Zhang T (2013) Loss of SHP-2 activity in CD4+ T cells promotes melanoma progression and metastasis. Sci Rep 3:2845. doi: 10.1038/srep02845 PubMedPubMedCentralGoogle Scholar
  3. 3.
    Lemeer S, Ruijtenbeek R, Pinkse MW, Jopling C, Heck AJ, den Hertog J, Slijper M (2007) Endogenous phosphotyrosine signaling in zebrafish embryos. Mol Cell Proteomics 6(12):2088–2099Google Scholar
  4. 4.
    Lemeer S, Jopling C, Naji F, Ruijtenbeek R, Slijper M, Heck AJ, den Hertog J (2007) Protein-tyrosine kinase activity profiling in knock down zebrafish embryos. PLoS One 2(7):e581CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Versele M, Talloen W, Rockx C, Geerts T, Janssen B, Lavrijssen T, King P, Gohlmann HW, Page M, Perera T (2009) Response prediction to a multitargeted kinase inhibitor in cancer cell lines and xenograft tumors using high-content tyrosine peptide arrays with a kinetic readout. Mol Cancer Ther 8(7):1846–1855CrossRefPubMedGoogle Scholar
  6. 6.
    Hilhorst R, Houkes L, Mommersteeg M et al (2013) Kinase activity profiling on peptide microarray. In: Bina M (ed) Gene regulation: methods and protocols, vol 977, Methods in molecular biology. Springer, Heidelberg, pp 259–271. doi: 10.1007/978-1-62703-284-1_21 CrossRefGoogle Scholar
  7. 7.
    van Ameijde J, Overvoorde J, Knapp S et al (2014) A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates. Anal Biochem 448:9–13CrossRefPubMedGoogle Scholar
  8. 8.
    van Ameijde J, Overvoorde J, Knapp S et al (2013) Real-time monitoring of the dephosphorylating activity of protein tyrosine phosphatases using microarrays with 3-nitrophosphotyrosine substrates. ChemPlusChem 78(11):1349–1357. doi: 10.1002/cplu.201300299 CrossRefGoogle Scholar
  9. 9.
    Hatta Y, Tsuchiya N, Matsushita M et al (1999) Identification of the gene variations in human CD22. Immunogenetics 49:280–286CrossRefPubMedGoogle Scholar
  10. 10.
    Hamblin AD, Hamblin TJ (2005) Functional and prognostic role of ZAP-70 in chronic lymphocytic leukaemia. Expert Opin Ther Targets 9:1165–1178CrossRefPubMedGoogle Scholar
  11. 11.
    Kalesh KA, Tan LP, Lu K, Gao L, Wang J, Yao SQ (2010) Peptide-based activity-based probes (ABPs) for target-specific profiling of protein tyrosine phosphatases (PTPs). Chem Commun 46:589–591. doi: 10.1039/B919744C CrossRefGoogle Scholar
  12. 12.
    Wisastra R, Poelstra K, Bischoff R, Maarsingh H, Haisma HJ, Dekker FJ (2011) Antibody-free detection of protein tyrosine nitration in tissue sections. ChemBioChem 12:2016–2020. doi: 10.1002/cbic.201100148 CrossRefPubMedGoogle Scholar
  13. 13.
    Yang J, Cheng Z, Niu T, Liang X, Zhao ZJ, Zhou GW (2000) Structural basis for substrate specificity of protein-tyrosine phosphatase SHP-1. J Biol Chem 275:4066–4071. doi: 10.1074/jbc.275.6.4066 CrossRefPubMedGoogle Scholar
  14. 14.
    Ames BN (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol 8:115–118. doi: 10.1016/0076-6879(66)08014-5 CrossRefGoogle Scholar
  15. 15.
    Hess HH, Derr JE (1975) Assay of inorganic and organic phosphorus in the 0.1–5 nanomole range. Anal Biochem 63(2):p607–p613CrossRefGoogle Scholar
  16. 16.
    Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89(11):4884–4887CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Hilhorst R, Houkes L, van den Berg A, Ruijtenbeek R (2009) Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A. Anal Biochem 387:150–161. doi: 10.1016/j.ab.2009.01.022 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Liesbeth Hovestad-Bijl
    • 1
  • Jeroen van Ameijde
    • 2
  • Dirk Pijnenburg
    • 1
  • Riet Hilhorst
    • 1
  • Rob Liskamp
    • 3
    • 4
  • Rob Ruijtenbeek
    • 1
  1. 1.PamGene International BV’s-HertogenboschThe Netherlands
  2. 2.Janssen Prevention CenterCrucell Holland BVLeidenThe Netherlands
  3. 3.School of ChemistryUniversity of GlasgowGlasgowUK
  4. 4.Medicinal Chemistry and Chemical BiologyUtrecht UniversityUtrechtThe Netherlands

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