Adenosine deaminase

O-(Pyridin-3-yl-methyl)hydroxylamine (PMOA), butyraldehyde, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (Tris), EDTA, DNase I (type IV, bovine pancreas), alkaline phosphatase (Escherichia coli), nuclease P1 (Penicillium citrinum), RNase A (bovine pancreas), RNase T1 (Aspergillus oryzae), and proteinase K (Tritirachium album) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphodiesterase I (Crotalus adamanteus venom) and adenosine deaminase were purchased from Worthington Biochemical Corp. (Lakewood, NJ). LC-MS grade solvents were purchased from Fisher Chemical Co. (Pittsburgh, PA). Puregene protein precipitation (PP) solution and DNeasy Blood & Tissue kit were purchased from Qiagen (Germantown, MD). N,N-Bis(2-chloroethyl)ethylamine hydrochloride was purchased from Aaron Chemistry Gmbh (Mittenwald, Germany). Pall 10 kDa filter with omega membrane, MWCO 10 kDa, was purchased from Sigma Aldrich (St. Louis, MO) All other chemicals were ACS grade and purchased from Sigma-Aldrich unless otherwise stated. Strata-X 33 μm Polymeric Reversed Phase 30 mg solid-phase extraction (SPE) cartridge was purchased from Phenomenex (Torrance, CA). Sola SCX cartridges were purchased from Thermo Scientific (Bellafonte, PA). MicroLiter autosampler vials with silanized glass inserts were purchased from Wheaton (Millville, NJ).

HEK293 cells expressing the recombinant human ARs were grown in DMEM supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO₂. Cells were collected after confluence by scraping into ice-cold PBS buffer. After homogenization and suspension, cells were centrifuged at 1,000g for 10 min, and the suspension was recentrifuged at 20,000g for 60 min at 4°C. The resultant pellets were resuspended in buffer containing 3 units/mL adenosine deaminase (Worthington Biochemical Corp., Lakewood, NJ) and incubated at 37°C for 30 min. The membrane preparation was split into aliquots which were kept at −80 °C until further use.