High‐throughput Phosphoproteomics Reveals the Role of Phosphorylation in Pluripotency and Phase Separation

Phosphorylation is a key post-translational modification involved in many cellular processes. Embryonic stem cells (ESCs), characterized by their self-renewal capacity and pluripotent differentiation potential, are widely used in studies of developmental biology and regenerative medicine. However, existing phosphoproteomic data for ESCs remain limited in throughput, restricting our understanding of phosphorylation-mediated regulatory mechanisms. In this study, we performed high-throughput phosphoproteomic profiling and identified 3711 phosphoproteins and 11,410 phosphosites. Integrated analyses showed that nearly half of the interacting proteins of the core pluripotency factors OCT4, SOX2, and NANOG are phosphorylated. Moreover, we found that phosphorylation is more prevalent in scaffold proteins involved in phase separation compared to clients and regulators, and is highly enriched in specific membraneless organelles, such as those in stress granules and Cajal bodies. Together, these findings provide a valuable resource for phosphoproteomics and offer important insights into the role of phosphorylation in pluripotency and phase separation.