Human iPSC-derived 2D cortical neurons and 3D cortical organoids show fetal brain proteomic similarity and distinct phosphoproteomic profiles.

Human iPSC-derived 2D cortical neurons and 3D cortical organoids (COs) are widely used to model early brain development in health and disease, but the molecular similarity of 2D and 3D models to human brain tissue at the protein and phosphorylation levels has not been systematically defined. Here, we performed paired global proteomic and phosphoproteomic profiling of 2D cortical neurons and 3D COs, and benchmarked both models against published human fetal brain, human adult frontal cortex, and mouse cortex reference datasets. Compared to mouse cerebral cortex, both 2D and 3D showed substantially higher proteomic similarity to human fetal brain. In contrast, phosphoproteomic profiles were poorly correlated between iPSC-derived models and adult human frontal cortex, and between human and mouse cortex, demonstrating that phosphorylation signaling is highly context- and developmental stage-dependent and not recapitulated by cross-species. Together, our findings establish a paired proteomic and phosphoproteomic benchmarking framework for human neuronal models, highlighting that 2D neurons and 3D COs are complementary platforms for neurodevelopmental research.