Quantification of 2′3′-cGAMP by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Quantification of 2′3′-cGAMP was performed by LC-MS/MS as described previously (30). Extraction of cGAMP from cell lysates was performed with acetonitrile/methanol/water (2/2/1, vol/vol/vol) buffer. 3′3′ cGAMP (500 pg, BioLog) was added to each sample as an internal standard. Extracts of untreated and Pf gDNA stimulated THP-1 cells were spiked with 3′3′-cGAMP and the levels of 2′3′-cGAMP (endogenous) and 3′3′-cGAMP internal standard were measured in parallel. As an additional control, extracts of untreated (medium control) THP-1 cells with an added spike of synthetic 2′3′-cGAMP (BioLog) were also prepared and used as positive controls.