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

Ole Pless, Elisabeth Kowenz-Leutz, Gunnar Dittmar, Achim Leutz*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

9 Citations (Scopus)

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/EBPÎ 2 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 3g-4 d to complete.

Original languageEnglish
Pages (from-to)359-364
Number of pages6
JournalNature Protocols
Volume6
Issue number3
DOIs
Publication statusPublished - Mar 2011
Externally publishedYes

Fingerprint

Dive into the research topics of 'A differential proteome screening system for post-translational modificationg-dependent transcription factor interactions'. Together they form a unique fingerprint.

Cite this