Characterization of the phospho-adhesome by mass spectrometry-based proteomics

Joseph Robertson, Jonathan D. Humphries, Nikki R. Paul, Stacey Warwood, David Knight, Adam Byron, Martin J. Humphries

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

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.

Original languageEnglish
Title of host publicationKinase Signaling Networks
EditorsAik-Choon Tan, Paul H. Huang
Place of PublicationNew York, NY
PublisherHumana Press, Inc
Chapter15
Pages235-251
Number of pages17
ISBN (Electronic)978-1-4939-7154-1
ISBN (Print)978-1-4939-7152-7
DOIs
Publication statusPublished - 21 Jul 2017

Publication series

NameMethods in Molecular Biology
Volume1636
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Journal Article

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