TY - JOUR
T1 - A differential proteome screening system for post-translational modificationg-dependent transcription factor interactions
AU - Pless, Ole
AU - Kowenz-Leutz, Elisabeth
AU - Dittmar, Gunnar
AU - Leutz, Achim
PY - 2011/3
Y1 - 2011/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=79952427477&partnerID=8YFLogxK
U2 - 10.1038/nprot.2011.303
DO - 10.1038/nprot.2011.303
M3 - Article
C2 - 21372816
AN - SCOPUS:79952427477
SN - 1754-2189
VL - 6
SP - 359
EP - 364
JO - Nature Protocols
JF - Nature Protocols
IS - 3
ER -