A study reports the first direct evidence of endogenous protein pyrophosphorylation in human cell lines using a tailored pyrophosphoproteomics workflow. The research identifies 148 pyrophosphosites across 71 human proteins, with NOLC1 and TCOF1 being the most heavily pyrophosphorylated. Pyrophosphorylation is mediated by inositol pyrophosphate (PP-InsP) messengers, which transfer their β-phosphoryl group to proteins in the presence of Mg²⁺ ions. Disruption of PP-InsP biosynthesis reduces rDNA transcription, likely by lowering pyrophosphorylation on regulatory proteins like NOLC1, TCOF1, and UBF1. The study highlights that pyrophosphorylation is a non-canonical phosphorylation mechanism, often occurring in acidic serine-rich stretches and localized to the nucleus and nucleolus. Pyrophosphorylation sites were validated using radiolabeled 5PP-InsP and confirmed through mass spectrometry. The research also shows that pyrophosphorylation is crucial for rRNA synthesis, as depletion of 5PP-InsP leads to reduced rDNA transcription. The study underscores the importance of pyrophosphorylation in cellular processes and suggests that it may serve as a regulatory mechanism in nucleolar functions. The findings provide a framework for future phosphoproteomic analyses and highlight the need for further investigation into the role of pyrophosphorylation in cellular signaling and regulation.A study reports the first direct evidence of endogenous protein pyrophosphorylation in human cell lines using a tailored pyrophosphoproteomics workflow. The research identifies 148 pyrophosphosites across 71 human proteins, with NOLC1 and TCOF1 being the most heavily pyrophosphorylated. Pyrophosphorylation is mediated by inositol pyrophosphate (PP-InsP) messengers, which transfer their β-phosphoryl group to proteins in the presence of Mg²⁺ ions. Disruption of PP-InsP biosynthesis reduces rDNA transcription, likely by lowering pyrophosphorylation on regulatory proteins like NOLC1, TCOF1, and UBF1. The study highlights that pyrophosphorylation is a non-canonical phosphorylation mechanism, often occurring in acidic serine-rich stretches and localized to the nucleus and nucleolus. Pyrophosphorylation sites were validated using radiolabeled 5PP-InsP and confirmed through mass spectrometry. The research also shows that pyrophosphorylation is crucial for rRNA synthesis, as depletion of 5PP-InsP leads to reduced rDNA transcription. The study underscores the importance of pyrophosphorylation in cellular processes and suggests that it may serve as a regulatory mechanism in nucleolar functions. The findings provide a framework for future phosphoproteomic analyses and highlight the need for further investigation into the role of pyrophosphorylation in cellular signaling and regulation.