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

ABSTRACT: 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 dete

SUBMITTER: Grawe C 

PROVIDER: S-EPMC10407929 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature