Reagents were as follows: 4-nitrobenzoic acid (4-NBA; Sigma Chemical Co., St. Louis, MO, USA), debrisoquine (as debrisoquine sulfate; Sigma Chemical Co.), acetonitrile (ACN; Fisher Optima grade, Fisher Scientific, Waltham, MA, USA), methanol (Fisher Optima grade, Fisher Scientific), chloroform (Fisher Optima grade, Fisher Scientific), water (Fisher Optima grade, Fisher Scientific), and PBS (Invitrogen, Carlsbad, CA, USA). Equipment and consumables were as follows: 15 ml centrifuge tubes (Corning, Corning, NY, USA), 2 ml microfuge tubes (Denville Scientific, Metuchen, NJ, USA), cell lifter (Thermo Fisher Scientific, Waltham, MA, USA), vacuum line for media and PBS aspiration, water bath (Thermo Fisher Scientific), bench-top centrifuge (Sorvall), microcentrifuge (Thermo Fisher Scientific), speed-vacuum concentrator (Savant), and an UPLC-quadrupole (Q)-TOF-LC-MS/MS system (Waters Corp., Milford, MA, USA).
The human breast cancer cell line MCF-7 was incubated as three biological replicates in 150 mm Petri dishes at 37°C under a gas phase of 95% air/5% CO2. The cells were maintained in MEM (Invitrogen), supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml; Gibco, UK). The cells were grown to approximately 80% confluence. The cells were washed with ice-cold PBS and detached using a cell scraper, transferred to a prechilled 15-ml conical tube, and kept on ice. A total cell count using a hemocytometer was performed, and approximately 10 million cells were aliquoted into tubes and spun down at 4°C. Excess PBS was removed by aspiration, and the cell pellets were suspended in 150 μL molecular biology grade water and lysed by two cycles of freeze thaw (30 s at −20°C, followed by 90 s in a 37°C water bath), followed by 30 s of sonication and stored on ice. Cell suspension (5 μL) was aliquoted and used for protein estimation by Bradford assay.
Methanol (600 μl) containing internal standards was added to the cell suspension, vortexed, and incubated on ice for 15 min, followed by addition of 600 μl chloroform. The tubes were vortexed and centrifuged at 13,000 rpm for 10 min at 15°C. The two phases were transferred to a fresh tube carefully avoiding the interface. Chilled ACN (600 μl) was added to each tube, vortexed, and incubated at −80°C for 2 h. The tubes were centrifuged at 13,000 rpm for 10 min at 4°C; the supernatant was transferred to fresh tubes and dried under vacuum. The two tubes for each sample were combined by resuspending in 150 μl 50% ACN/water, followed by UPLC-TOF-MS analysis.
Each sample (5 μl) was injected onto a reverse-phase 50 × 2.1 mm Acquity 1.7-μm C18 column (Waters Corp.) using an Acquity UPLC system (Waters Corp.) with a gradient mobile phase consisting of 2% ACN in water containing 0.1% formic acid (Solvent A) and 2% water in ACN containing 0.1% formic acid (Solvent B) and resolved for 10 min at a flow rate of 0.5 mL/min. The gradient consisted of 100% A for 0.5 min with a ramp of curve 6–100% B from 0.5 min to 10 min. The column eluent was introduced directly into the mass spectrometer by electrospray. MS was performed on a Q-TOF Premier (Waters Corp.), operating in a negative-ion (ESI−) or positive-ion (ESI+) electrospray ionization mode with a capillary voltage of 3200 V and a sampling cone voltage of 20 V in negative mode and 35 V in positive mode. The desolvation gas flow was set to 800 L/h, and the temperature was set to 350°C. The cone gas flow was 25 L/h, and the source temperature was 120°C. Accurate mass was maintained by introduction of LockSpray interface of sulfadimethoxine (311.0814 [M+H]+ or 309.0658 [M−H]−) at a concentration of 250 pg/μL in 50% ACN/water and a rate of 150 μL/min. Data were acquired in centroid mode from 50 to 850 mass-to-charge ratio (m/z) in MS scanning. Centroided and integrated MS data from the UPLC-TOF-MS were processed to generate a multivariate data matrix using MarkerLynx (Waters Corp.).