biostudies-literatureGrawe CEuropean Research CouncilStichting Saxum VolutumKWF Kankerbestrijding2683-2693https://www.ebi.ac.uk/biostudies/studies/S-EPMC10407929biostudies-literatureUnknown22(8)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. MS¹-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 deteJournal of proteome researchDetermining DNA-Protein Binding Affinities and Specificities from Crude Lysates Using a Combined SILAC/TMT Labeling Strategy.PMC10407929771059Hernandez-Quiles MJansen PWTCGrawe CVermeulen MBrimmers AfalseDetermining DNA-Protein Binding Affinities and Specificities from Crude Lysates Using a Combined SILAC/TMT Labeling Strategy.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. MS¹-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 dete2023-01-01T00:00:00Z2023 Aug2025-04-26T06:32:00.442Z2025-04-06T11:54:32.437ZS-EPMC104079293746616410.1021/acs.jproteome.3c00248