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Quantitative Phosphoproteomics to Study cAMP Signaling

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cAMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2483))

Abstract

Cyclic adenosine monophosphate (cAMP) signaling activates multiple downstream cellular targets in response to different stimuli. Specific phosphorylation of key target proteins via activation of the cAMP effector protein kinase A (PKA) is achieved via signal compartmentalization. Termination of the cAMP signal is mediated by phosphodiesterases (PDEs), a diverse group of enzymes comprising several families that localize to distinct cellular compartments. By studying the effects of inhibiting individual PDE families on the phosphorylation of specific targets it is possible to gain information on the subcellular spatial organization of this signaling pathway.

We describe a phosphoproteomic approach that can detect PDE family-specific phosphorylation changes in cardiac myocytes against a high phosphorylation background. The method combines dimethyl labeling and titanium dioxide–mediated phosphopeptide enrichment, followed by tandem mass spectrometry.

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Acknowledgments

This work was supported by the British Heart Foundation (PG/10/75/28537 and RG/17/6/32944) and the BHF Centre of Research Excellence, Oxford (RE/13/1/30181).

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Authors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK

    Katharina Schleicher & Manuela Zaccolo

  2. Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, UK

    Svenja Hester

  3. Department of Biochemistry, University of Oxford, Oxford, UK

    Svenja Hester

  4. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Monika Stegmann

Authors
  1. Katharina Schleicher
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  2. Svenja Hester
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  3. Monika Stegmann
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  4. Manuela Zaccolo
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Editor information

Editors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK

    Manuela Zaccolo

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Schleicher, K., Hester, S., Stegmann, M., Zaccolo, M. (2022). Quantitative Phosphoproteomics to Study cAMP Signaling. In: Zaccolo, M. (eds) cAMP Signaling. Methods in Molecular Biology, vol 2483. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2245-2_18

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  • DOI: https://doi.org/10.1007/978-1-0716-2245-2_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2244-5

  • Online ISBN: 978-1-0716-2245-2

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