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Human Pluripotent Stem Cells pp 297–312Cite as

Quantitative Proteome and Phosphoproteome Analysis of Human Pluripotent Stem Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 767))

Abstract

Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regulating signaling pathways in biological processes. Global analysis of dynamic changes in protein phosphorylation is, therefore, key to understanding signaling at the system level. Here, we describe a generic mass spectrometry (MS)-based phosphoproteomics strategy applied to monitor phosphorylation dynamics after bone morphogenetic protein 4 (BMP4)-induced differentiation of human embryonic stem cells (hESCs). Our method combines the use of strong cation exchange (SCX) and titanium dioxide (TiO2) for phosphopeptide enrichment, high-resolution MS for peptide and protein identification, and stable isotope labeling by amino acids in cell culture (SILAC) for quantification. This approach allows us to identify thousands of phosphorylation sites and profile their relative abundance during differentiation. This systems-biology-based approach provides new insights into how human pluripotent stem cells exit the pluripotent state.

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Acknowledgments

Parts of the work described here were supported by the Bsik programs “Dutch Platform for Tissue Engineering,” “Stem Cells in Development and Disease,” the Netherlands Proteomic Center, and the FP6 EU Program Heart Development and Heart Repair. We also acknowledge the contributions from the Mummery and Heck groups, especially Dennis van Hoof, Stefan Braam, Martijn Pinkse, Shabaz Mohammed, and Jeroen Krijgsveld.

Author information

Authors and Affiliations

  1. Bijvoet Center for Biomolecular Research, Biomolecular Mass Spectrometry and Proteomics Group, Utrecht, The Netherlands

    Javier Muñoz & Albert J. R. Heck

  2. Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Javier Muñoz & Albert J. R. Heck

Authors
  1. Javier Muñoz
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  2. Albert J. R. Heck
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Corresponding author

Correspondence to Albert J. R. Heck .

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Editors and Affiliations

  1. Research Institute, CHOC Human Neural Stem Cell Resource, Children's Hospital of Orange County, S. Main Street 455, Orange, 92868-3874, California, USA

    Philip H. Schwartz

  2. Beckman Research Institute, Center for Applied Technology Developmen, City of Hope, East Duarte Road 1500, Duarte, 91010, California, USA

    Robin L. Wesselschmidt

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Muñoz, J., Heck, A.J.R. (2011). Quantitative Proteome and Phosphoproteome Analysis of Human Pluripotent Stem Cells. In: Schwartz, P., Wesselschmidt, R. (eds) Human Pluripotent Stem Cells. Methods in Molecular Biology, vol 767. Humana Press. https://doi.org/10.1007/978-1-61779-201-4_22

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  • DOI: https://doi.org/10.1007/978-1-61779-201-4_22

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-200-7

  • Online ISBN: 978-1-61779-201-4

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