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Innate Antiviral Immunity pp 153–166Cite as

cGAMP Quantification in Virus-Infected Human Monocyte-Derived Cells by HPLC-Coupled Tandem Mass Spectrometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1656))

Abstract

Upon virus infection, cells of the innate immune system such as dendritic cells and macrophages can mount type I interferon (IFN-I) responses that restrict viral dissemination. To inform host cells of virus infection, detection of cytosolic DNA is one important mechanism. Inappropriate sensing of endogenous DNA and subsequent induction of IFN-I responses can also cause autoimmunity, highlighting the need to tightly regulate DNA sensing. The cyclic GMP-AMP synthase (cGAS) was recently identified to be the major sensor of cytosolic DNA that triggers IFN-I expression. Upon DNA binding, cGAS synthesizes the second messenger cyclic guanosine-adenosine monophosphate (cGAMP) that induces IFN-I expression by the activation of the stimulator of interferon genes (STING). Notably, cGAMP does not only act in infected cells, but can also be relocated to noninfected bystander cells to there trigger IFN-I expression. Thus, direct quantification of cGAMP in cells of the innate immune system is an important approach to study where, when, and how DNA is sensed and IFN-I responses are induced. Here, we describe a method that allows specific quantification of cGAMP from extracts of virus-infected human myeloid cells by HPLC-coupled tandem mass spectrometry.

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Acknowledgments

We gratefully acknowledge the skillful technical assistance of Annette Garbe. This study was supported by funding from the Helmholtz Virtual Institute (VH-VI-424 Viral Strategies of Immune Evasion, VISTRIE) and from the Helmholtz-Alberta Initiative, Infectious Diseases Research (HAI-IDR SO-073) to UK.

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Authors and Affiliations

  1. Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover Medical School, Feodor-Lynen-Str. 7, Hannover, 30625, Germany

    Jennifer Paijo & Ulrich Kalinke

  2. Research Core Unit Metabolomics, Hannover Medical School, Hannover, 30625, Germany

    Volkhard Kaever

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  1. Jennifer Paijo
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  2. Volkhard Kaever
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  3. Ulrich Kalinke
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Correspondence to Ulrich Kalinke .

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  1. Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada

    Karen Mossman

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Paijo, J., Kaever, V., Kalinke, U. (2017). cGAMP Quantification in Virus-Infected Human Monocyte-Derived Cells by HPLC-Coupled Tandem Mass Spectrometry. In: Mossman, K. (eds) Innate Antiviral Immunity. Methods in Molecular Biology, vol 1656. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7237-1_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7237-1_9

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