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LC-MS/MS in Proteomics pp 187–204Cite as

Proteome-Wide Quantitation by SILAC

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 658))

Abstract

Ongoing improvements in instrumentation, fractionation techniques, and enrichment procedures have dramatically increased the coverage of the proteome achievable via LC-MS/MS-based methodologies, opening the call for approaches to quantitatively assess differences at a proteome-wide scale. Stable isotope labeling by amino acids in cell culture (SILAC) has emerged as a powerful and versatile approach for proteome-wide quantitation by mass spectrometry. SILAC utilizes the cells’ own metabolism to incorporate isotopically labeled amino acids into its proteome which can be mixed with the proteome of unlabeled cells and differences in protein expression can easily be read out by comparing the abundance of the labeled versus unlabeled proteins. SILAC has been applied to numerous different cell lines and the technique has been adapted for a wide range of experimental procedures. In this chapter we provide detailed procedure for performing SILAC-based experiment for proteome-wide quantitation, including a protocol for optimizing SILAC labeling. We also provide an update on the most recent developments of this technique.

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Acknowledgements

We would like to thank all members of the Center for Experimental BioInformatics (CEBI) for useful discussions, especially Dr. Irina Kratchmarova for the critical reading of the chapter. The research leading to these results has received funding from the European Commission’s 7th Framework Programme (grant agreement HEALTH-F4-2008-201648/PROSPECTS), the Danish Natural Science Research Council and the Lundbeck Foundation.

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Authors and Affiliations

  1. Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

    Kristoffer T.G. Rigbolt & Blagoy Blagoev

Authors
  1. Kristoffer T.G. Rigbolt
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  2. Blagoy Blagoev
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Editors and Affiliations

  1. Bart’s and the London School of Medicine, Institute of Cancer, Queen Mary, Universtiy of London, Charterhouse Square, London, EC1M 6BQ, United Kingdom

    Pedro R. Cutillas

  2. , EGA Institute for Women's Health, University College London, Gower Street, London, WC1E 6BT, United Kingdom

    John F. Timms

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Rigbolt, K.T., Blagoev, B. (2010). Proteome-Wide Quantitation by SILAC. In: Cutillas, P., Timms, J. (eds) LC-MS/MS in Proteomics. Methods in Molecular Biology, vol 658. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-780-8_11

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  • DOI: https://doi.org/10.1007/978-1-60761-780-8_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-779-2

  • Online ISBN: 978-1-60761-780-8

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