Principle of the DNA–protein interaction screen. Proteomes are metabolically labeled with 2H4-lysine (Lysine-d4) to allow discrimination based on differences in peptide mass (4 Da). Biotinylated (Bio) DNA molecules bearing functional elements (e.g., potential TF binding sites or methylated CpGs) are synthesized and immobilized on streptavidin magnetic beads. Control columns are designed to lack the functional element of interest (e.g., point mutations in cis-elements or nonmethylated CpGs). Nuclear extracts from unlabeled (Lysine-d0) and labeled cells are prepared and subjected to DNA affinity chromatography, followed by the release of DNA–protein complexes by restriction enzyme digestion. After in-gel digestion with trypsin, differentially labeled forms of lysine containing tryptic peptides are detected by mass spectrometry (MS). Peptides originating from proteins specifically recognizing functional elements will have a larger peak intensity of the lysine-d4 (Kd4) form. Unspecific interaction partners will have 1:1 ratios between both isotopic forms.
