Reversible protein phosphorylation is a central signaling mechanism in eukaryotes. Although mass-spectrometry-based phosphoproteomics has become routine, identification of non-canonical phosphorylation has remained a challenge. Here we report a tailored workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing, to our knowledge, the first direct evidence of endogenous protein pyrophosphorylation. We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. Detection was consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). When the biosynthesis of PP-InsPs was perturbed, proteins expressed in this background exhibited no signs of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1 and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation and should be considered in future phosphoproteomic analyses.

可逆的蛋白质磷酸化是真核生物的核心信号传导机制。尽管基于质谱的磷酸化蛋白质组学已成为常规方法，但非经典磷酸化的鉴定仍然是一个挑战。在这里，我们报告了一个定制的工作流程，用于检测和可靠地分配两种人类细胞系中的蛋白质焦磷酸化，据我们所知，提供了内源蛋白质焦磷酸化的第一个直接证据。我们手动验证了 71 种人类蛋白质的 148 个焦磷酸位点，其中焦磷酸化最严重的是核仁蛋白质 NOLC1 和 TCOF1。检测结果与之前有关肌醇焦磷酸 (PP-InsPs) 修饰的生化证据一致。当 PP-InsPs 的生物合成受到干扰时，在此背景下表达的蛋白质没有表现出焦磷酸化的迹象。 PP-InsP 生物合成的破坏也显着减少了 rDNA 转录，这可能是通过降低调节蛋白 NOLC1、TCOF1 和 UBF1 的焦磷酸化来实现的。总体而言，蛋白质焦磷酸化作为非经典磷酸化的原型出现，应在未来的磷酸蛋白质组学分析中予以考虑。