Protein–DNA Arrays as Tools for Detection of Protein–Protein Interactions by Mass Spectrometry

Authors

  • Dr. Lars Gogolin,

    1. Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching (Germany)
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  • Dr. Hendrik Schroeder,

    1. Technische Universität Dortmund, Department of Chemistry, Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
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  • Prof. Dr. Aymelt Itzen,

    1. Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching (Germany)
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  • Prof. Dr. Roger S. Goody,

    1. Max-Planck Institute of Molecular Physiology, Otto-Hahn Strasse 11, 44227 Dortmund (Germany)
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  • Prof. Dr. Christof M. Niemeyer,

    1. Technische Universität Dortmund, Department of Chemistry, Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
    2. Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen (Germany)
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  • Prof. Dr. Christian F. W. Becker

    Corresponding author
    1. Institute of Biological Chemistry, Universität Wien, Währinger Strasse 38, 1090 Wien (Austria)
    2. Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching (Germany)
    • Institute of Biological Chemistry, Universität Wien, Währinger Strasse 38, 1090 Wien (Austria)
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Abstract

Analysis of multiple protein–protein interactions using microarray technology remains challenging, and site-specific immobilization of functional proteins is a key step in these approaches. Here we establish the efficient synthesis of protein–DNA conjugates for several members of a small family of GTPases. The family of Rab/Ypt GTPases is intimately involved in vesicular trafficking in yeast and serves as a model for the much larger group of analogous human proteins, the Rab protein family, with more than 60 members. The Ypt–DNA hybrid molecules described here are used for DNA-directed immobilization on glass- and silica-based microarrays. Methods for the detection of protein–DNA conjugates, as well as approaches for nucleotide exchange and distinguishing between GDP- and GTP-bound Ypts on microarrays, are reported. The high specificity of different Rab/Ypt-effector interactions, which also depends on the bound nucleotide, is shown by fluorescence readout of microarrays. Furthermore, initial experiments demonstrate that direct readout by mass spectrometry can be achieved with commercially available instruments. These developments will significantly contribute to the elucidation of complex transport networks in eukaryotic cells.

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