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

[Image: see text] In recent years, quantitative mass spectrometry-based interaction proteomics technology has proven very useful in identifying specific DNA–protein interactions using single pull-downs from crude lysates. Here, we applied a SILAC/TMT-based higher-order multiplexing approach to develop an interaction proteomics workflow called Protein–nucleic acid Affinity and Specificity quantification by MAss spectrometry in Nuclear extracts or PASMAN. In PASMAN, DNA pull-downs using a concentration range of specific and control DNA baits are performed in SILAC-labeled nuclear extracts. MS(1)-based quantification to determine specific DNA–protein interactions is then combined with sequential TMT-based quantification of fragmented SILAC peptides, allowing the generation of Hill-like curves and determination of apparent binding affinities. We benchmarked PASMAN using the SP/KLF motif and further applied it to gain insights into two CGCG-containing consensus DNA motifs. These motifs are recognized by two BEN domain-containing proteins, BANP and BEND3, which we find to interact with these motifs with distinct affinities. Finally, we profiled the BEND3 proximal proteome, revealing the NuRD complex as the major BEND3 proximal protein complex in vivo. In summary, PASMAN represents, to our knowledge, the first higher-order multiplexing-based interaction proteomics method that can be used to decipher specific DNA–protein interactions and their apparent affinities in various biological and pathological contexts.