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Shotgun Proteomics pp 251–263Cite as

Identification of DNA Damage Checkpoint-Dependent Protein Interactions in Saccharomyces cerevisiae Using Quantitative Mass Spectrometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1156))

Abstract

The DNA damage checkpoint (DDC) is an evolutionarily conserved signaling pathway that is crucial to maintain genomic integrity. In response to DNA damage, DDC kinases are rapidly activated and phosphorylate an elaborate network of substrates involved in multiple cellular processes. An important role of the DDC response is to assemble protein complexes. However, for most of the DDC substrates, how the DDC-dependent phosphorylation modulates their network of interactions remains to be established. Here, we present a protocol for the identification of DDC-dependent protein–protein interactions based on Stable Isotope Labeling of Amino acids in Cell culture (SILAC) followed by affinity-tagged protein purification and quantitative mass spectrometry analysis. Based on a model study using Saccharomyces cerevisiae, we provide a method that can be generally applied to study the role of kinases in mediating protein–protein interactions.

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Acknowledgments

We thank Beatriz S. Almeida for technical support. M.B.S. is supported by grants from the National Institute of Health (R01-GM097272) and American Cancer Society (RSG-11-146-01-DMC) and F.M.B.d.O. is supported by a Cornell Fleming Research Fellowship.

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Authors and Affiliations

  1. Institute of Biophysics, Federal University of Rio de Janeiro, Av Carlos Chagas Filho, 373 CCS Bloco G, Rio de Janeiro, RJ, 21941-902, Brazil

    Francisco M. Bastos de Oliveira

  2. Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, 339 Weill Hall, Ithaca, NY, 14853-7202, USA

    Marcus B. Smolka

Authors
  1. Francisco M. Bastos de Oliveira
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  2. Marcus B. Smolka
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Corresponding author

Correspondence to Marcus B. Smolka .

Editor information

Editors and Affiliations

  1. Ludwig-Maximilians-University, Munich, Germany

    Daniel Martins-de-Souza

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de Oliveira, F.M.B., Smolka, M.B. (2014). Identification of DNA Damage Checkpoint-Dependent Protein Interactions in Saccharomyces cerevisiae Using Quantitative Mass Spectrometry. In: Martins-de-Souza, D. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 1156. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0685-7_17

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  • DOI: https://doi.org/10.1007/978-1-4939-0685-7_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0684-0

  • Online ISBN: 978-1-4939-0685-7

  • eBook Packages: Springer Protocols

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