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SILAC-Based Temporal Phosphoproteomics

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Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1188))

Abstract

In recent years, thanks to advances in Mass Spectrometry (MS)-based quantitative proteomics, studies on signaling pathways have moved from a detailed description of individual components to system-wide analysis of entire signaling cascades, also providing spatio-temporal views of intracellular pathways. Quantitative proteomics that combines stable isotope labeling by amino acid in cell culture (SILAC) with enrichment strategies for post-translational modification-bearing peptides and high-performance tandem mass spectrometry represents a powerful and unbiased approach to monitor dynamic signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated tyrosines by immunoaffinity and then further enriched for phosphorylated serine/threonine peptides by strong cation exchange in combination with titanium dioxide-beads chromatography. Analysis of enriched peptides on Orbitrap-based MS results in comprehensive and accurate reconstruction of temporal changes of signaling networks.

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Acknowledgements

The authors thank all members of the Department of Proteomics at the Novo Nordisk Foundation Center for Protein Research for fruitful discussion. The work was supported by the seventh framework program of the European Union (Contract no. 262067—PRIME-XS). The NNF Center for Protein Research was supported by a generous donation from the Novo Nordisk Foundation. Dr. Chiara Francavilla was supported by Marie Curie and EMBO Long-Term postdoctoral fellowships. Dr. Blagoev was supported by the Lundbeck Foundation.

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Authors and Affiliations

  1. Department of Proteomics, Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Blegdamsvej 3B, 2200, Copenhagen, Denmark

    Chiara Francavilla, Omid Hekmat & Jesper V. Olsen

  2. Department of Biochemistry and Molecular Biology, Center for Experimental BioInformatics, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark

    Blagoy Blagoev

Authors
  1. Chiara Francavilla
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  2. Omid Hekmat
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  3. Blagoy Blagoev
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  4. Jesper V. Olsen
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Corresponding authors

Correspondence to Blagoy Blagoev or Jesper V. Olsen .

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Editors and Affiliations

  1. Institute of Biology II, University of Freiburg, Freiburg, Germany

    Bettina Warscheid

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Francavilla, C., Hekmat, O., Blagoev, B., Olsen, J.V. (2014). SILAC-Based Temporal Phosphoproteomics. In: Warscheid, B. (eds) Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Methods in Molecular Biology, vol 1188. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1142-4_10

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  • DOI: https://doi.org/10.1007/978-1-4939-1142-4_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1141-7

  • Online ISBN: 978-1-4939-1142-4

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