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

  • Jennifer Paijo
  • Volkhard Kaever
  • Ulrich KalinkeEmail author
Part of the Methods in Molecular Biology book series (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.

Key words

cGAMP cGAS Macrophage Dendritic cell Type I interferon HPLC Tandem mass spectrometry 

Notes

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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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jennifer Paijo
    • 1
  • Volkhard Kaever
    • 2
  • Ulrich Kalinke
    • 1
    Email author
  1. 1.Institute for Experimental Infection Research, TWINCORECentre for Experimental and Clinical Infection Research, Hannover Medical SchoolHannoverGermany
  2. 2.Research Core Unit MetabolomicsHannover Medical SchoolHannoverGermany

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