Proteome-wide quantification of inositol pyrophosphate-protein interactions.

Inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are highly phosphorylated signaling molecules involved in diverse cellular processes. To resolve discrete signaling events mediated by these structurally related metabolites, a mass spectrometry-based approach was developed to derive apparent binding constants on a proteome-wide scale. The method employs chemically synthesized affinity reagents for inositol hexakisphosphate (InsP6) and the inositol pyrophosphates 1PP-InsP5, 5PP-InsP5, and 1,5(PP)2-InsP4 (InsP8). Concentration-dependent affinity enrichment combined with tandem mass tag (TMT) labeling enabled identification and quantification of ligand-protein interactions for hundreds of proteins from mammalian cell lysates. Biochemical and functional validation of selected targets demonstrated engagement with endogenous ligands. Comparison of enrichment conditions revealed a strong dependence of PP-InsP binding on Mg2+ ions. Additionally, gene ontology analysis linked PP-InsP interactors to nuclear and nucleolar RNA processing, and subsequent analyses could identify several pyrophosphorylation sites, previously uncharacterized. In summary, these datasets provide valuable resources for exploring PP-InsP-dependent signaling pathways across biological systems.