Proteome-wide quantification of inositol pyrophosphate-protein interactions

Abstract

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.