15 deoxy δ12 14pgj2

Prostaglandins in cell supernatants were extracted and analyzed according to Idborg et al. [19 (link)]. Working in duplicates, 450 μl of samples were spiked with 50 μl deuterated internal standards of 6-keto-PGF_1α, PGF_2α, PGE₂, PGD₂, TxB₂, and 15-deoxy-Δ12,14PGJ₂ (Cayman Chemical Company) and made acidic with 500 μl 0.2 % formic acid (FA) in Milli-Q. The samples were loaded on Oasis HLB 1 cc 30 mg cartridge (Waters Corporation, MA, USA) that had been preconditioned with methanol and 0.05 % FA in Milli-Q. Prostaglandins were eluted in 1 ml methanol and evaporated to dryness under vacuum. Samples were stored at −20 °C until resuspended in 50 μl 7 % acetonitrile prior to analysis with LC-MS/MS. The extracted prostaglandins were quantified using a triple quadrupole mass spectrometer (Acquity TQ Detector, Waters Corporation) equipped with a Waters 2795 HPLC (Waters Corporation). Separation was performed on a 100 × 2.0 mm Synergi 2.5 μm Hydro-RP 100 Å column (Phenomenex, CA, USA) with a 45-min stepwise linear gradient (10–90 %) of 0.05 % FA in acetonitrile as mobile phase B and Milli-Q as mobile phase A. Individual prostaglandins were detected in multiple reaction monitoring mode. Data were analyzed using MassLynx Software, version 4.1, with internal standard calibration and quantification to external standard curves.

PGD₂, 13,14-dihydro-15-keto-PGD₂, PGJ₂, Δ¹²-PGJ₂, Δ¹²-PGD₂, 15-deoxy-Δ^12,14-PGJ₂, 15-deoxy-Δ^12,14-PGD₂, 9α,11β-PGF₂ were purchased from Cayman Chemicals (Biomol GmBH, Hamburg, Germany). Fevipiprant (GST0000013789) was provided by Novartis Pharma AG (Basel, Switzerland). Reagents were dissolved in sterile-filtered Hybri-Max dimethylsulfoxide (DMSO, Sigma-Aldrich, Taufkirchen, Germany).

Appropriate dilutions of total membrane fraction protein in duplicates were made in activity assay buffer containing 0.1M potassium phosphate buffer pH 7.5 (0.1% Triton X-100, 1% glycerol and 2.5mM GSH) to obtain a final concentration of 300μg protein/ml. These were incubated with PGH₂ (final concentration 10μM), GSH (2.5mM final concentration) for 60 seconds at room temperature. After incubation, the reaction was immediately stopped by adding 400μl of stop solution containing 25mM FeCl₂ and 50mM citric acid. The remaining PGH₂ is converted mainly to 12‐ HHT and MDA by the ferric ions. A deuterated internal standard of PGE₂, PGD₂, PGF2_α, TXB₂, 6-keto-PGF_1α, 13,14-dihydro-15-keto-PGE₂ and 15-deoxy-Δ 12, 14 PGJ₂ (Cayman Chemicals, Michigan, USA) was added to each sample. The WT and variant enzyme E. coli membrane fractions were analyzed. Denatured (boiled for 10 minutes) samples were incorporated as negative controls.