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Here, we applied quantitative proteomic and phosphoproteomic analyses to profile the neuronal 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 phosphorylated peptides, which revealed molecular signatures during the differentiation of iPSC-derived neurons. Proteomic data highlighted distinct changes in mitochondrial pathways throughout the course of differentiation, whereas phosphoproteomic data revealed specific regulatory dynamics in GTPase-mediated signaling pathways and microtubule proteins. Additionally, phosphosite dynamics were not correlated to changes in protein abundance, particularly in processes related to axon functions and RNA transport. We measured the dynamic changes in kinases that are critical for neuronal development and maturation and developed an interactive web app to visualize the temporal landscape dynamics of protein and phosphosite abundance. By establishing baselines of proteomic and phosphoproteomic profiles for neuronal differentiation, this dataset is a valuable resource for future research into neuronal development and neurodegenerative diseases using this reference iPSC line.<\/jats:p>","DOI":"10.1126\/scisignal.adx8680","type":"journal-article","created":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T18:58:13Z","timestamp":1771959493000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":3,"title":["Temporal proteomic and phosphoproteomic dynamics during neuronal differentiation in the reference iPSC line KOLF2.1J"],"prefix":"10.1126","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5053-4850","authenticated-orcid":true,"given":"Ying","family":"Hao","sequence":"first","affiliation":[{"name":"Center for Alzheimer\u2019s and Related Dementias (CARD), National Institute on Aging and National Institute of Neurological Disorders and Stroke, 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