A proteomics protocol to identify stimulation-induced binding partners dependent on a specific gene in mammalian cells

Some protein-protein interactions are induced by different kinds of stimulation and are dependent on specific genes. To identify these interaction partners, we present a protocol which utilizes affinity purification of Flag-tagged protein complexes followed by mass-spectrometry-based proteomics to compare stimulation-induced interactomes between wild-type and CRISPR-Cas9-mediated knockout cells. The candidates of interest are identified using bioinformatic analyses and verified by biochemical approaches. This protocol is highly versatile and applies to a variety of cells and different types of stimulation. For complete details on the use and execution of this protocol, please refer to (Zhu et al., 2021).