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

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Item Type:	Article
Title:	A differential proteome screening system for post-translational modification-dependent transcription factor interactions
Creators Name:	Pless, O., Kowenz-Leutz, E., Dittmar, G. and Leutz, A.
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/EBPbeta 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.
Keywords:	Arginine, Methylation, Molecular Sequence Data, Peptide Library, Peptides, Protein Binding, Post-Translational Protein Processing, Proteins, Proteome, Proteomics, Transcription Factors
Source:	Nature Protocols
ISSN:	1745-2473
Publisher:	Nature Publishing Group
Volume:	6
Number:	3
Page Range:	359-364
Date:	February 2011
Official Publication:	https://doi.org/10.1038/nprot.2011.303
PubMed:	View item in PubMed

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