This pipeline presents a refined approach for deriving personalized neurobiological insights from iPSC-derived neurospheres. By employing Tandem Mass Tag (TMT) labeling, we optimized sample pooling and multiplexing for robust comparative analysis across experimental conditions, maximizing data yield per sample. Through single-patient-derived neurospheres-composed of neural progenitor cells, early neurons, and radial glia-this study explores proteomic profiling to mirror the cellular complexity of neurodevelopment more accurately than traditional 2D cultures. Given their enhanced relevance, these 3D neurospheres serve as a valuable model for elucidating neurogenesis, differentiation, and neuropathological mechanisms, contributing to the advancement of in vitro neural models and reducing dependency on animal models. SIGNIFICANCE: This study evaluates ten protein extraction protocols using TMT 10-plex labeling to optimize proteomic analysis from single neurospheres. It compares cost, protein yield, and the ability to detect differentially expressed proteins, identifying methods like SPEED and S-Trap as efficient for high-throughput studies, while FASP excels in peptide yield. TMT labeling enhances protein identification, particularly for low-abundance proteins, and allows pre-fractionation to maximize analysis from limited samples. However, challenges such as limited PTM analysis and the potential loss of minor proteins highlight the importance of selecting protocols based on specific research goals. This work contributes to optimizing proteomic workflows for in vitro neural models, advancing single-cell analysis with minimal reliance on animal models.

Guillaume Nugue

Michele Martins

Gabriela Vitória

Beatriz Luzia De Mello Lima Guimaraes

Mauricio Quiñones-Vega

Stevens Rehen

Marilia Z Guimarães

Magno Junqueira

Journal of Proteomics

蛋白质组学杂志面向蛋白质组学、生物标志物发现、蛋白质分析、植物蛋白质组学、微生物和动物蛋白质组学、人体研究、质谱组织成像、非常规和非模型生物蛋白质组学领域的蛋白质科学家和分析化学家，和蛋白质生物信息学。该杂志欢迎新领域和即将到来的领域的论文，如代谢组学、基因组学、系统生物学、毒理学组学、药物蛋白质组学。蛋白质组学杂志统一了基础科学家和临床医生，并包括转化研究。欢迎对评论、网络研讨会和主题问题提出建议。

The Journal of Proteomics is a journal dedicated to proteomics, biomarker discovery, protein analysis, plant proteomics, microbiome and animal proteomics, human studies, mass spectrometry imaging, as well as unconventional and non-model organism proteomics. This journal serves protein scientists, analytical chemists, and proteomics bioinformaticians. We welcome research papers from emerging and cutting-edge fields, including metabolomics, genomics, systems biology, toxicology, and drug proteomics. The Journal of Proteomics aims to bridge the gap between basic scientists and clinicians, encompassing translational research. We also welcome suggestions for reviews, webinars, and thematic issues.

《Journal of Proteomics》是一本专注于蛋白质组学、生物标志物发现、蛋白质分析、植物蛋白质组学、微生物和动物蛋白质组学、人体研究、质谱组织成像以及非常规和非模型生物蛋白质组学的期刊。该杂志旨在服务于蛋白质科学家、分析化学家和蛋白质生物信息学家。我们欢迎来自新兴领域和前沿领域的研究论文，包括代谢组学、基因组学、系统生物学、毒理学组学和药物蛋白质组学。《Journal of Proteomics》致力于促进基础科学家与临床医生之间的交流，并涵盖转化研究。我们也欢迎对评论、网络研讨会和主题问题的建议。

Category	Quartile
生物学	2
生物学, 生化研究方法	2

Optimized pipeline for personalized neurobiological insights from single patient-derived Neurospheres

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Journal Info

Category	Quartile
BIOCHEMICAL RESEARCH METHODS	2