RT Journal Article
SR Electronic
T1 Proteome-wide quantification of inositol pyrophosphate-protein interactions
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2024.07.31.606004
DO 10.1101/2024.07.31.606004
A1 Richter, Annika
A1 Ruwolt, Max
A1 Lampe, Sarah
A1 Liu, Fan
A1 Furkert, David
A1 Fiedler, Dorothea
YR 2024
UL http://biorxiv.org/content/early/2024/07/31/2024.07.31.606004.abstract
AB Inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs) constitute a group of highly phosphorylated molecules that are involved in many cellular signaling processes. To characterize discrete signaling events of these structurally closely related molecules, a mass spectrometry approach was developed to derive apparent binding constants for these ligands on a proteome-wide scale. The method employed a series of chemically synthesized, biotinylated affinity reagents for inositol hexakisphosphate (InsP6), and the inositol pyrophosphates 1PP-InsP5, 5PP-InsP5 and 1,5(PP)2-InsP4 (also termed InsP8). Application of these affinity reagents at different concentrations, in combination with tandem mass tag (TMT) labeling, provided binding data for thousands of proteins from a mammalian cell lysate. Investigation of different enrichment conditions, where Mg2+ ions were either available or not, showcased a strong influence of Mg2+ on the protein binding capacities of PP-InsPs. Gene ontology analysis closely linked PP-InsP-interacting proteins to RNA processing in the nucleus and nucleolus. Subsequent data analysis enabled a targeted search for protein pyrophosphorylation among PP-InsP interactors, and identified four new pyrophosphorylated proteins. The data presented here constitute a valuable resource for the community, and application of the method reported here to other biological contexts will enable the exploration of PP-InsP dependent signaling pathways across species.Competing Interest StatementThe authors have declared no competing interest.