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This chapter presents an optimized method for isolating synaptic vesicles (SVs) from neurospheres derived from human induced pluripotent stem cells (hiPSCs). The protocol begins with neurosphere cultivation to achieve mature neurons, which is essential for the functional studies of neuronal activity. Following this, neurosphere-derived synaptosomes are isolated, and SVs are enriched through differential centrifugation. The method culminates in the proteomic analysis of SVs using nano-liquid chromatography coupled with high-resolution tandem mass spectrometry (nanoLC-MS/MS), providing a detailed proteome profile of the isolated vesicles. This protocol can contribute to the understanding of SV molecular heterogeneity and the mechanisms of neurotransmitter uptake and release and be applied to the field of research in neurological and neuropsychiatric disorders.

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Isolating Synaptic Vesicles from Neurospheres for Proteomics

  • 实验方案

  • 发表日期：

    2025年

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  • Caroline Brandão-Teles,
  • Giuliana S. Zuccoli,
  • Marcelo Ganzella,
  • Victor Corasolla Carregari,
  • Linda Olsthoorn,
  • Érica de Almeida Duque,
  • Carolina Demarchi Munhoz,
  • Reinhard Jahn,
  • Daniel Martins-de-Souza,
  • Fernanda Crunfli

摘要

This chapter presents an optimized method for isolating synaptic vesicles (SVs) from neurospheres derived from human induced pluripotent stem cells (hiPSCs). The protocol begins with neurosphere cultivation to achieve mature neurons, which is essential for the functional studies of neuronal activity. Following this, neurosphere-derived synaptosomes are isolated, and SVs are enriched through differential centrifugation. The method culminates in the proteomic analysis of SVs using nano-liquid chromatography coupled with high-resolution tandem mass spectrometry (nanoLC-MS/MS), providing a detailed proteome profile of the isolated vesicles. This protocol can contribute to the understanding of SV molecular heterogeneity and the mechanisms of neurotransmitter uptake and release and be applied to the field of research in neurological and neuropsychiatric disorders.