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Proteomic Analysis of Human Neural Stem Cell Differentiation by SWATH-MS

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Embryonic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2520))

Abstract

The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.

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Acknowledgments

This work was supported by the Czech Ministry of Education, Youth and Sports project InterCOST (LTC18079) under CellFit COST Action (CA16119), the Charles University, projects GA UK No. 1767518 and No. 1460217, and the Czech Science Foundation (19-01747S).

Author information

Authors and Affiliations

  1. Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Libechov, Czech Republic

    Jirina Tyleckova, Jakub Cervenka, Ievgeniia Poliakh, Jaromir Novak, Katerina Vodickova Kepkova, Helena Kupcova Skalnikova & Petr Vodicka

  2. Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic

    Jakub Cervenka, Ievgeniia Poliakh & Jaromir Novak

Authors
  1. Jirina Tyleckova
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  2. Jakub Cervenka
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  3. Ievgeniia Poliakh
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  4. Jaromir Novak
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  5. Katerina Vodickova Kepkova
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  6. Helena Kupcova Skalnikova
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  7. Petr Vodicka
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Corresponding author

Correspondence to Petr Vodicka .

Editor information

Editors and Affiliations

  1. Ottawa, ON, Canada

    Kursad Turksen

1 Electronic Supplementary Material

Data 1

MSstats_Input_Example (CSV 69246 kb)

Data 2

MSstats_script_example (R 8 kb)

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Tyleckova, J. et al. (2022). Proteomic Analysis of Human Neural Stem Cell Differentiation by SWATH-MS. In: Turksen, K. (eds) Embryonic Stem Cell Protocols . Methods in Molecular Biology, vol 2520. Humana, New York, NY. https://doi.org/10.1007/7651_2022_462

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  • DOI: https://doi.org/10.1007/7651_2022_462

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2436-4

  • Online ISBN: 978-1-0716-2437-1

  • eBook Packages: Springer Protocols

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