Communications
DOI: 10.1002/anie.200701601
Microarrays
A Microarray Strategy for Mapping the Substrate Specificity of Protein
Tyrosine Phosphatase**
Maja Köhn, Marta Gutierrez-Rodriguez, Pascal Jonkheijm, Stefan Wetzel, Ron Wacker,
Hendrik Schroeder, Heino Prinz, Christof M. Niemeyer, Rolf Breinbauer, Stefan E. Szedlacsek,
and Herbert Waldmann*
Reversible phosphorylation of tyrosine residues in proteins is
involved in regulating numerous cellular events, such as cell
growth, cell differentiation, cell-cycle regulation, cellular
signal transduction, cell adhesion, and the immune
response.[1] Phosphorylation is controlled by protein tyrosine
kinases (PTKs) and protein tyrosine phosphatases (PTPs).
Although PTKs have been studied extensively, knowledge
about PTP substrate specificities is still limited.[2] The
corresponding data could be used to create artificial sub-
strates for kinetic studies,[3] to design inhibitors[4] leading to
drug candidates and tools to investigate PTP pathways in
cells, and might even give hints for the identification of
natural substrates.[5] Several peptide-based approaches have
been reported for mapping PTP substrate specificity.[6]
the rapid screening of multiple substrate candidates in
parallel. Microarray techniques offer the opportunity for the
fast and efficient screening of a large number of phospho-
peptides, as demonstrated for kinase substrate mapping.[8] Up
to now however, microarrays have been applied in only a very
few cases for substrate mapping of PTPs.[9]
We report here the construction of phosphotyrosine
(pTyr) peptide microarrays and their application in the
mapping of the substrate specificity of two prototypical
PTPs: PTPm and PTP1B. Our methodology consists of five
steps (A–E in Figure 1). First, a library of individual pTyr
peptides is synthesized on the solid phase (A). Individual
peptides are subsequently immobilized onto glass surfaces in
a spatially addressable manner (B, C). For this covalent
immobilization, we[10] made use of the chemoselective Stau-
dinger ligation between an azide and a phosphine previously
reported by the research groups of Raines and Bertozzi.[11]
The pTyr peptide microarrays are then incubated with the
Solution-phase methods are usually employed for the
detection of the substrates,[7] but they often do not allow for
[*] Dr. M. Köhn, Dr. M. Gutierrez-Rodriguez, Dr. P. Jonkheijm,
Dipl.-Chem. S. Wetzel, Priv.-Doz. H. Prinz, Prof. R. Breinbauer,
Prof. H. Waldmann
PTP of interest (D), washed, treated with a fluorescently
[12]
labeled anti-pTyr antibody
and washed again (E). In
Department of Chemical Biology
Max-Planck Institute für molekulare Physiologie
Otto Hahn Strasse 11, 44227 Dortmund(Germany)
and
Fachbereich Chemie
Organische Chemie
Universität Dortmund
44227 Dortmund(Germany)
Fax: (+49)231-133-2499
E-mail: herbert.waldmann@mpi-dortmund.mpg.de
general, preferred substrates of the PTP display no or lower
fluorescence signals relative to a control array, whereas no
change in the signal intensity indicates unsuitable substrates.
A library of a tetrapeptide, a hexapeptide, and 46
pentapeptides was synthesized that covered a variety of
different types of amino acids in different positions (see the
Supporting Information, p. 1). The peptides were prepared
following a strategy recently reported by us[10] which employs
a resin equipped with a sulfamylbutyryl linker.[12] Release
from the resin introduces a hexylchain-linked azide that
allows immobilization without any further activation step.
The performance of the pTyr peptide microarray was
tested using PTP1B, a well-studied PTP in regard to substrate
specificity.[3,6] The amino acid sequence of peptide 1[19]
(Figure 2) is a known substrate for PTP1B (Km = 3.2 mm).
Since PTP1B, in general, prefers acidic side chains,[13] peptide
Dr. R. Wacker
Chimera Biotec GmbH
Emil-Figge-Strasse 76a, 44227 Dortmund(Germany)
Dr. H. Schroeder, Prof. C. M. Niemeyer
Fachbereich Chemie
Biologisch-Chemische Mikrostrukturtechnik
Universität Dortmund
Otto-Hahn Strasse 6, 44227 Dortmund(Germany)
2
[19] (Figure 2) was selected from the library to determine the
Prof. S. E. Szedlacsek
Department of Enzymology
Institute of Biochemistry
Splaiul Independentei 296, 060031 Bucharest (Romania)
preference of substrate 1 over 2 to be dephosphorylated.
Peptides 1 and 2 were spotted onto phosphane-modified glass
slides (1 mm, 250 pL/spot).[12] The phosphanes were attached
to dendrimer-coated glass slides that are known to enhance
surface coverage.[14] To minimize interactions of the PTP as
well as the peptides with the surface we introduced two
aminohexyl linkers, that is, one linker between the dendrimer
and the phosphane and another one between the peptides and
the azide.[12] Incubation of the arrays with various concen-
trations of PTP1B and subsequent treatment with a conjugate
[**] This work was supportedby the Max-Planck Gesellschaft, the
research program “Molecular Basics of Biosciences” of the
University of Dortmund, the Fonds der Chemischen Industrie, and
the Zentrum für Angewandte Chemische Genomik. P.J. thanks the
Alexander von Humboldt Stiftung and S.W. thanks Novartis for a
scholarship.
Supporting information for this article is available on the WWW
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 7700 –7703