Quantification of nucleotides (ATP, ADP, AMP, and ZMP) in aortic smooth muscle cells using LC-MS/MS MRM

Human aortic smooth muscle cells, vascular cell basal medium (Cat # PCS-100-030), and vascular smooth muscle cell growth kit (Cat # PCS-100-042) were purchased from ATCC (Manassas, VA). Aortic smooth muscle cells (passages 3 to 6) were maintained in culture with vascular cell basal medium containing vascular smooth muscle cell growth kit and antibiotic/antimycotic solution in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. After the attainment of confluence (∼6–7 days), smooth muscle cells were trypsinized, centrifuged, and 3 million cells were seeded on to 150 mm petri dishes each, as described previously [16]. Subconfluent smooth muscle cells were deprived of growth supplements for 2 days followed by incubation with Krebs–Henseleit bicarbonate (KHB) buffer for 90 min. Subsequently, smooth muscle cells were exposed to vehicle control or 1 mM AICAR for 30 min. Smooth muscle cells were then washed twice with ice-cold PBS and collected in 1 mL ice-cold PBS. The cell suspension was centrifuged at 1,000×g for 5 min at 4 °C to pellet the cells. After discarding the supernatant, the cell pellet was snap-frozen using liquid N₂ and stored at −80 °C until analysis. On the day of nucleotide extraction, 200 µl of 5% ice-cold perchloric acid was added to the cell pellet to lyse the cells and sample was further centrifuged at 10,000×g for 3 min at 4 °C to remove the acid-insoluble material. Perchloric acid in the collected supernatant was extracted by three washes with 10% excess volume of a 1:1 mixture of tri-n-octylamine and 1,1,2-trichlorotrifluroethane. The nucleotides remaining in the aqueous phase were then analyzed using liquid chromatography/tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) on a 4000 QTRAP LC/MS/MS system (Applied Biosystems, Carlsbad, CA) [9]. The concentrations of nucleotides (ATP, ADP, AMP, and ZMP) in the samples were calculated using the standard curve prepared from serial dilutions of the respective nucleotide stock solution (1 mM). In brief, the samples and standards were run on an Amide XBridge HPLC column (Cat # 186,004,860; 3.5 µM particle size; 2.1 mm inner diameter×100 mm length, Waters, Milford, MA) using buffer A (20 mM ammonium hydroxide and 20 mM ammonium acetate in 5% acetonitrile, pH 9.0) and buffer B (100% acetonitrile) at a flow rate of 0.3 mL/min for 10 min. The mobile phase consisted of isocratic elusion with 20% buffer B. The detailed specifications of MRM transitions for nucleotides are described in Table 1.

MRM transitions for nucleotides (ATP, ADP, AMP, and ZMP).

CE, collision energy; DP, declustering potential; EP, entrance potential; CXP, collision cell exit potential.