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Kinase Signaling Networks pp 235–251Cite as

Characterization of the Phospho-Adhesome by Mass Spectrometry-Based Proteomics

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1636))

Abstract

Integrin adhesion receptors engage with their extracellular matrix (ECM) ligands, initiating intracellular signaling pathways that regulate a range of fundamental cell functions. Protein kinases and phosphatases play an integral role in integrin adhesion-mediated signaling. However, until recently, knowledge of the phosphorylation sites regulated downstream of integrin ligation was limited to candidate-based approaches and did not support a system-level understanding of the molecular mechanisms through which ECM engagement influences cell behavior. Here, we describe a mass spectrometry (MS)-based phosphoproteomic protocol that enables the global characterization of phosphorylation-based signaling networks activated by integrin-mediated adhesion. To analyze specifically integrin-proximal signaling, the phosphoproteomic workflow involves the affinity-based isolation and analysis of integrin-associated complexes (IACs) rather than proteins solubilized from whole-cell lysates , which are typically used for global phosphoproteomic studies. The detection of phosphorylation sites from IAC proteins was optimized at various stages of the workflow, including IAC isolation, proteolytic digestion, and MS-based data acquisition strategies. The protocol permits the identification and quantification of IAC components by both Western blotting and MS. Notably, compared to phosphoproteomic analyses of cell lysates, the workflow described here enables an improved detection of phosphorylation sites from well-defined IAC proteins, including many known components of the signaling pathways activated by adhesion to the ECM.

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Acknowledgments

This work was supported by the Wellcome Trust (grant 092015 to M.J.H.), a Wellcome Trust Institutional Strategic Support Fund award (grant 097820 to the University of Manchester) and a Biotechnology and Biological Sciences Research Council studentship (to J.R.). The authors would also like to thank J.N. Selley for bioinformatic support.

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Author notes

  1. Joseph Robertson and Jonathan D. Humphries contributed equally to this work.

Authors and Affiliations

  1. Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

    Joseph Robertson, Jonathan D. Humphries, Nikki R. Paul, Adam Byron & Martin J. Humphries

  2. Department of Molecular Microbiology, Oslo University Hospital, 0027, Oslo, Norway

    Joseph Robertson

  3. CRUK Beatson Institute, Glasgow, G61 1BD, UK

    Nikki R. Paul

  4. Biological Mass Spectrometry Core Facility, Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK

    Stacey Warwood & David Knight

  5. Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, EH4 2XR, UK

    Adam Byron

Authors
  1. Joseph Robertson
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  2. Jonathan D. Humphries
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  3. Nikki R. Paul
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  4. Stacey Warwood
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  5. David Knight
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  6. Adam Byron
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  7. Martin J. Humphries
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Corresponding author

Correspondence to Martin J. Humphries .

Editor information

Editors and Affiliations

  1. Division of Medical Oncology, University of Colorado, Aurora, Colorado, USA

    Aik-Choon Tan

  2. Protein Networks Laboratory, Institute of Cancer Research, London, United Kingdom

    Paul H. Huang

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Robertson, J. et al. (2017). Characterization of the Phospho-Adhesome by Mass Spectrometry-Based Proteomics. In: Tan, AC., Huang, P. (eds) Kinase Signaling Networks. Methods in Molecular Biology, vol 1636. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7154-1_15

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  • DOI: https://doi.org/10.1007/978-1-4939-7154-1_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7152-7

  • Online ISBN: 978-1-4939-7154-1

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