Hao Ying, Li Ziyi, Lara Erika, Ramos Daniel M, Santiana Marianist, Jin Benjamin, Epstein Jacob, Camacho Jasmin, Carmiol Nicole, Kowal Isabelle, Jarreau Paige, Weller Cory A, Klaisner Sydney, Screven Laurel A, Pantazis Caroline B, Nalls Mike A, Narayan Priyanka, Ferrucci Luigi, Singleton Andrew B, Ward Michael E, Cookson Mark R, Qi Yue Andy
bioRxiv. 2025 Mar 26:2025.03.25.645331. doi: 10.1101/2025.03.25.645331.
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.
Temporal dynamics of proteome and phosphoproteome profiles in KOLF2.1J iPSC derived neurons.Phosphoproteomics highlights GTPase signaling and microtubule regulation in neuronal differentiation.Kinome mapping reveals a shift in kinase activity patterns from early to late differentiation.Shinyapp for visualizing the trajectory of protein and phosphosite expression during neuronal differentiation.
诱导多能干细胞（iPSC）衍生的神经元已成为研究神经元发育和神经退行性疾病的强大模型。尽管转录组学和成像技术已被用于表征神经元发育特征，但蛋白质和翻译后修饰（PTM）的综合数据集尚不可得。在这里，我们应用定量蛋白质组学和磷酸蛋白质组学来分析KOLF2.1J iPSC系的分化情况，该系是iPSC神经退行性疾病倡议（iNDI）项目的首个参考系。我们开发了一种自动化工作流程，能够实现蛋白质和磷酸化蛋白质的高覆盖率富集。我们的结果揭示了iPSC衍生神经元分化过程中蛋白质组和磷酸蛋白质组层面的分子特征。蛋白质组学数据突出了分化过程中线粒体途径的明显变化，而磷酸蛋白质组学揭示了GTPase信号通路和微管蛋白中的特定调控动态。此外，磷酸化位点动态与蛋白质表达呈现出不一致的趋势，特别是在与轴突功能和RNA运输相关的过程中。此外，我们绘制了对神经元发育和成熟至关重要的激酶动态变化。我们开发了一个交互式网络应用程序（https://niacard.shinyapps.io/Phosphoproteome/）来可视化蛋白质和磷酸化位点表达的时间动态景观。通过建立神经元分化的蛋白质组和磷酸蛋白质组图谱基线，该数据集为未来使用这个参考iPSC系研究神经元发育和神经退行性疾病提供了宝贵资源。
KOLF2.1J iPSC衍生神经元中蛋白质组和磷酸蛋白质组图谱的时间动态。磷酸蛋白质组学突出了神经元分化中的GTPase信号传导和微管调节。激酶组图谱揭示了从早期到晚期分化过程中激酶活性模式的转变。用于可视化神经元分化过程中蛋白质和磷酸化位点表达轨迹的Shinyapp。
