| Assay Method Information | |
| | Rapidfire Mass Spectroscopy Assay |
| Description: | Table 2 and 3: Compounds were resuspended in 10 mM stock concentration using DMSO and tested to determine their IC50 values against h-cGAS in 384-well polypropylene plates using RapidFire 365 mass spectrometry (RF-MS). The final concentration of full-length h-cGAS, dsDNA, ATP, and GTP were 100 nM, 25 nM, 100 μM, and 100 μM, respectively. The reaction buffer was composed of 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 μM ZnCl2 and 0.01% Tween-20. Reaction solutions of 20 μl were incubated for 7 h at room temperature (RT) and stopped by addition of 60 μl of 0.5% (v/v) formic acid per well followed by RF-MS analysis. An aqueous solvent of 5 mM ammonium acetate, pH 10 was used for loading/washing process. An organic solvent comprising 5 mM ammonium acetate, pH 10 in 50% water, 25% acetone, and 25% acetonitrile was used for elution of the analytes. About 35 μl of each sample was aspirated from a 384-well plate and separated using a Graphitic carbon Type D cartridge. The sample loaded onto cartridge was then washed for 4 s at 1.5 ml min−1 using the aqueous solvent. ATP, GTP, and cGAMP were eluted for 5 s using the organic solvent at a flow rate of 1.5 ml min−1 followed by re-equilibration with the aqueous solvent for 5 s at a flow rate of 1.5 ml min−1. The samples were analysed using a negative ionization mode in the mass spectrometer, with a gas temperature of 350° C., nebulizer pressure of 35 psi, and gas flow rate of 15 L min−1. The acquisition range was between 300 and 800 m/z for all the chromatograms and the molecular masses of the detected peaks were: ATP: 505.9835, GTP: 521.9854, and cGAMP: 673.0906. The area under the curve (AUC) of the extracted ion counts for each analyte was calculated using the Agilent RapidFire Integrator software. |
| Affinity data for this assay | |
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