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A differential proteome screening system for post-translational modification–dependent transcription factor interactions

Nature Protocols volume 6, pages 359364 (2011) | Download Citation

Abstract

Post-translational modifications (PTMs) of transcription factors alter interactions with co-regulators and epigenetic modifiers. For example, members of the C/EBP transcription factor family are extensively methylated on arginine and lysine residues in short, conserved, modular domains, implying modification-dependent cofactor docking. Here we describe array peptide screening (APS), a systematic and differential approach to detect PTM-dependent interactions in the human proteome using chemically synthesized, biotinylated peptides coupled to fluorophore-labeled streptavidin. Peptides with and without a modified residue are applied in parallel to bacterial expression libraries in an arrayed format. Interactions are detected and quantified by laser scanning to reveal proteins that differentially bind to nonmodified or modified peptides. We have previously used this method to investigate the effect of arginine methylation of C/EBPβ peptides. The method enables determination of PTM-dependent transcription factor interactions, quantification of relative binding affinities and rapid protein classification, all independently of the transactivation potential of peptides or cellular abundance of interactors. The protocol provides a cost-effective alternative to mass spectrometric approaches and takes 3–4 d to complete.

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Acknowledgements

We thank K.W. Friedrich and U. Englisch from LI-COR Biosciences for technical support and discussions. We thank M. Knoblich for protein purification for subsequent mass spectrometric analysis. O.P. is supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to A.L. (LE 770/4-1). E.K.-L., G.D. and A.L. are supported by institutional funds from the Helmholtz Association.

Author information

Affiliations

  1. Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.

    • Ole Pless
    • , Elisabeth Kowenz-Leutz
    • , Gunnar Dittmar
    •  & Achim Leutz
  2. Max-Delbrück-Center for Molecular Medicine, Mass Spectrometry Core Facility, Berlin, Germany.

    • Gunnar Dittmar
  3. Humboldt University of Berlin, Berlin, Germany.

    • Achim Leutz

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Contributions

O.P. and A.L. designed experiments, and O.P. and E.K.-L. performed experiments and analyzed data. G.D. performed mass spectrometric analysis and data evaluation. O.P. and A.L. prepared the manuscript. A.L. supervised the work.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Achim Leutz.

Supplementary information

Word documents

  1. 1.

    Supplementary Table 1

    Differential interaction partners on UniPEx library, part 1

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DOI

https://doi.org/10.1038/nprot.2011.303

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