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Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) pp 227–244Cite as

Analyzing the Protein Assembly and Dynamics of the Human Spliceosome with SILAC

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

Abstract

Quantitative mass spectrometry has become an indispensable tool in proteomic studies. Numerous methods are available and can be applied to approach different issues. In most studies these issues include the quantitative comparison of different cell states, the identification of specific interaction partners or determining degrees of posttranslational modification. In this chapter we describe a SILAC-based quantification in order to analyze dynamic protein changes during the assembly of the human spliceosome on a pre-mRNA in vitro. We provide protocols for assembly of spliceosomes on pre-mRNA (including generation of pre-mRNAs and preparation of nuclear extracts), quantitative mass spectrometry (SILAC labeling, sample preparation), and data analysis to generate timelines for the dynamic protein assembly.

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Acknowledgements

We thank Klaus Hartmuth for fruitful discussions and Uwe Plessmann and Johanna Lehne for excellent technical assistance.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, 12 Mansfield Road, OX1 3TA, Oxford, UK

    Carla Schmidt

  2. Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany

    Monika Raabe & Henning Urlaub

  3. Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany

    Reinhard Lührmann

  4. Bioanalytics, Department of Clinical Chemistry, University Medical Center, Robert-Koch-Strasse 40, Göttingen, Germany

    Henning Urlaub

Authors
  1. Carla Schmidt
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  2. Monika Raabe
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  3. Reinhard Lührmann
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  4. Henning Urlaub
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Corresponding author

Correspondence to Henning Urlaub .

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Editors and Affiliations

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

    Bettina Warscheid

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Schmidt, C., Raabe, M., Lührmann, R., Urlaub, H. (2014). Analyzing the Protein Assembly and Dynamics of the Human Spliceosome with SILAC. 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_16

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  • DOI: https://doi.org/10.1007/978-1-4939-1142-4_16

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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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