Temporal dynamics of proteome and phosphorproteome during neuronal differentiation in the reference KOLF2.1J iPSC line

Hao, Y.; Li, Z.; Lara, E.; Ramos, D. M.; Santiana, M.; Jin, B.; Epstein, J.; Camacho, J.; Carmiol, N.; Kowal, I.; Jarreau, P.; Weller, C. A.; Klaisner, S.; Screven, L. A.; Pantazis, C. B.; Nalls, M. A.; Narayan, P.; Ferrucci, L.; Singleton, A. B.; Ward, M. E.; Cookson, M. R.; Qi, Y. A.

doi:10.1101/2025.03.25.645331

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bioRxiv · 10.1101/2025.03.25.645331

Temporal dynamics of proteome and phosphorproteome during neuronal differentiation in the reference KOLF2.1J iPSC line

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Abstract

Induced pluripotent stem cell (iPSC)-derived neurons have emerged as a powerful model to investigate both neuronal development and neurodegenerative diseases. Although transcriptomics and imaging have been applied to characterize neuronal development signatures, comprehensive datasets of protein and post-translational modifications (PTMs) are not readily available. Here, we applied quantitative proteomics and phosphoproteomics to profile the differentiation of the KOLF2.1J iPSC line, the first reference line of the iPSC Neurodegenerative Disease Initiative (iNDI) project. We developed an automated workflow enabling high-coverage enrichment of proteins and phosphoproteins. Our results revealed molecular signatures across proteomic and phosphoproteomic landscapes during differentiation of iPSC-derived neurons. Proteomic data highlighted distinct changes in mitochondrial pathways throughout the course of differentiation, while phosphoproteomics revealed specific regulatory dynamics in GTPase signaling pathways and microtubule proteins. Additionally, phosphosite dynamics exhibited discordant trends compared to protein expression, particularly in processes related to axon functions and RNA transport. Furthermore, we mapped the kinase dynamic changes that are critical for neuronal development and maturation. We developed an interactive Web app (https://niacard.shinyapps.io/Phosphoproteome/) to visualize temporal landscape dynamics of protein and phosphosite expression. By establishing baselines of proteomic and phosphoproteomic profiles for neuronal differentiation, this dataset offers a valuable resource for future research into neuronal development and neurodegenerative diseases using this reference iPSC line. HighlightsO_LITemporal dynamics of proteome and phosphoproteome profiles in KOLF2.1J iPSC derived neurons. C_LIO_LIPhosphoproteomics highlights GTPase signaling and microtubule regulation in neuronal differentiation. C_LIO_LIKinome mapping reveals a shift in kinase activity patterns from early to late differentiation. C_LIO_LIShinyapp for visualizing the trajectory of protein and phosphosite expression during neuronal differentiation. C_LI

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Hao, Y., Li, Z., Lara, E., Ramos, D. M., Santiana, M., Jin, B., Epstein, J., Camacho, J., Carmiol, N., Kowal, I., Jarreau, P., Weller, C. A., Klaisner, S., Screven, L. A., Pantazis, C. B., Nalls, M. A., Narayan, P., Ferrucci, L., Singleton, A. B., Ward, M. E., Cookson, M. R., Qi, Y. A.. 2025-03-26. Temporal dynamics of proteome and phosphorproteome during neuronal differentiation in the reference KOLF2.1J iPSC line. https://doi.org/10.1101/2025.03.25.645331

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