Fig. 1. Validation of RapidFire Mass Spectrometry System (RF-MSS) cyclase assay and high throughput screening conditions.

An example of a RF-MS chromatogram showing (a) the product cAMP as extracted ion intensity (EIC) × 10³, and (b) substrate ATP as EIC × 10⁴. Peaks derive from increasing input ATP (in mM) recorded at time “0” (black) and after 180 minutes incubation with sAC (red). (c) Human sAC activity measured using RF-MSS as a function of substrate of ATP-Mg²⁺ for 120 min in the presence of excess MgCl₂ (20 mM). Shown are curves for Mg²⁺ alone (red dots), Mg²⁺/40 mM HCO₃⁻ (green squares), Mg²⁺/10 mM Ca²⁺ (blue triangles), and Mg²⁺/40 mM HCO₃⁻/10 mM Ca²⁺ (purple triangles). Determinations are representative of at least two independent experiments and curves are nonlinear fits generated by Prism. (d–f) RF-MSS screen assaying sAC in the presence of 1 mM ATP/5 mM MgCl₂/5 mM CaCl₂/40 mM NaHCO₃. (d) Comparison of active (black dots) and denatured (open squares) sAC enzyme: Z’ score = 0.7. (e) LOPAC library (1280 compounds) pilot screen (black); DMSO control (red); denatured sAC protein (blue). (f) Results of screening LOPAC library twice. LOPAC compounds (gray); DMSO control (green square); denatured sAC protein (red). (R² = 0.81).