Determining DNA-Protein Binding Affinities and Specificities from Crude Lysates Using a Combined SILAC/TMT Labeling Strategy

In recent years,quantitative mass spectrometry-based interactionproteomics technology has proven very useful in identifying specificDNA-protein interactions using single pull-downs from crudelysates. Here, we applied a SILAC/TMT-based higher-order multiplexingapproach to develop an interaction proteomics workflow called Protein-nucleicacid Affinity and Specificity quantification by MAss spectrometryin Nuclear extracts or PASMAN. In PASMAN, DNA pull-downs using a concentrationrange of specific and control DNA baits are performed in SILAC-labelednuclear extracts. MS1-based quantification to determinespecific DNA-protein interactions is then combined with sequentialTMT-based quantification of fragmented SILAC peptides, allowing thegeneration of Hill-like curves and determination of apparent bindingaffinities. We benchmarked PASMAN using the SP/KLF motif and furtherapplied it to gain insights into two CGCG-containing consensus DNAmotifs. These motifs are recognized by two BEN domain-containing proteins,BANP and BEND3, which we find to interact with these motifs with distinctaffinities. Finally, we profiled the BEND3 proximal proteome, revealingthe NuRD complex as the major BEND3 proximal protein complex in vivo.In summary, PASMAN represents, to our knowledge, the first higher-ordermultiplexing-based interaction proteomics method that can be usedto decipher specific DNA-protein interactions and their apparentaffinities in various biological and pathological contexts.