SEQUENCE-SPECIFIC CAPTURE OF PROTEIN-DNA COMPLEXES FOR MASS SPECTROMETRIC PROTEIN IDENTIFICATION

摘要

Characterization of proteins associated with genetic locations-promoters, enhancers, silencers, insulators-is critical to learn how proteins work in concert to regulate transcription. Methods using an antibody (ChIP) or tag (FLAG, TAP) to pull down a protein of interest and analyze its associated factors (proteins, ligands, DNA) are popular today. Although these strategies provide insight into regulatory networks, they require a working antibody or recombinantly tagged protein and fall short of completely identifying unknown proteins bound to a genomic site. To identify proteins endogenously associated with DNA, we have developed a strategy called GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, protein-DNA complexes are enriched using sequence-specific DNA hybridization and the associated proteins are identified using mass spectrometry.