Affinity enrichment and identification of inositol poly- and pyrophosphate interactomes

This protocol describes an affinity enrichment approach from mammalian cell extracts to identify protein binding partners of inositol hexakisphosphate (InsP₆) and 5-diphosphoinositol pentakisphosphate (5PP-InsP₅), two important eukaryotic metabolites. The interactomes are annotated using mass spectrometry-based proteomics, and comparison against a control resin can uncover hundreds of protein targets. Quantitative analysis of InsP₆- versus 5PP-InsP₅-binding proteins highlights specific protein-ligand interactions. The approach is applicable to different cells and organisms and will contribute to a mechanistic understanding of inositol poly- and pyrophosphate signaling. For complete details on the use and execution of this protocol, please refer to Furkert et al. (2020).