Extensive protein pyrophosphorylation revealed in human cell lines

Reversible protein phosphorylation is a central signaling mechanism in eukaryotes, but detection of non-canonical phosphorylations remains challenging. The authors report a tailored mass spectrometry workflow to detect and unambiguously assign protein pyrophosphorylation (conversion of phosphoserine to pyrophosphoserine) in two human cell lines, providing, to their knowledge, the first direct evidence of endogenous protein pyrophosphorylation. They manually validated 148 pyrophosphosites across 71 proteins, with nucleolar proteins NOLC1 and TCOF1 most heavily modified. Detection was consistent with prior biochemical evidence linking modification to inositol pyrophosphates (PP-InsPs). Perturbation of PP-InsP biosynthesis abolished detectable pyrophosphorylation in expressed proteins, and disruption of PP-InsPs significantly reduced rDNA transcription, potentially via reduced pyrophosphorylation of NOLC1, TCOF1 and UBF1. The study establishes protein pyrophosphorylation as an archetype of non-canonical phosphorylation to be considered in future phosphoproteomics.