Waters acquity uhplc system

Analysis of cellular fractions were performed on an AB Sciex 4000 QTRAP® Mass Spectrometer(SCIEX, USA) connected with an Waters ACQUITY UHPLC system (Waters Co., USA), using a X Bridge^TM BEH C18 analytical column (1.7 μm 2.1 × 50 mm; Waters, Torrance, CA). A linear gradient mobile phase composed of 0.1% formic acid water (solvent A) and acetonitrile (solvent B) was mixed according to the following gradient program: 0–5 min (20–55% B); 5–12 min (55–65% B); 12–13 min (65–20% B). The injection volume was 10 μl and flow rate was 0.4 ml/min.
For our final method, MS parameters were as follows: Ionspray voltage, 4000 V; source temperature, 400 °C; Gas 1/2, 30 psi; curtain gas, 30 psi; collision energy, 30 V; dwell time, 100 ms. Precursor-to-product ion transition of m/z 403.3 → 373.2 for nobiletin and m/z 271.0 → 150.8 for naringenin (internal standard) were used for multiple reaction monitoring (MRM).

Purified shell-cytochromes were diluted to ~5 μM (based on the extinction coefficient from the Soret band absorbance) in NH₄Ac. Five microliters of sample was injected onto a Hypersil Gold cyanopropyl guard column (1 × 10 mm) (Thermo Scientific, Massachusetts, USA) using a Waters Acquity UHPLC system (Waters, Massachusetts, USA). A binary gradient flowing at 0.1 ml min⁻¹ to elute the protein was programmed as follows: initial conditions 98% A (water + 0.15% formic acid) / 2% B (acetonitrile), held for 5 min to wash away salts and buffer; a linear ramp to 75% B over 5 min (from 5 to 10 min after loading); hold at 75% B for 2 min (until 12 min after load); return to starting conditions of 98% A / 2% B at 12.01 min and hold 3 min (until 15 min after load) to re-equilibrate the column for the next injection. Proteins were analyzed on a Waters G2-XS quadrupole-time-of-flight mass spectrometer using electrospray ionization operating in positive ion mode and scanning a mass range of m/z 200–2,000 with 1 scan per second. Capillary voltage was 3 kV, sample cone voltage was 35 V, source temperature was 100°C, desolvation temperature was 350°C and desolvation gas flow was 600 L hr⁻¹. Elution Peaks at 7–9 min were integrated and the molecular weight spectra were deconvoluted using the MaxEnt 1 algorithm in the Waters Masslynx software package.

FFA containing 12 or more carbon atoms were analyzed using a Waters ACQUITY-UHPLC system (Waters Corp, Milford, USA) coupled to AB SCIEX Triple Q TOF 5600 plus System (AB SCIEX, Framingham, USA) operated in negative ion mode as previously described (18 (link)) with slight modifications. The separation was performed on a 2.1 × 100 mm ACQUITY 1.8 µm T3 column (Waters, Milford, MA, USA) and the mobile phase consisted of 6.5 mM ammonium bicarbonate in water (A) and 6.5 mM ammonium bicarbonate in 95% MeOH and water (B). The gradient elution started at 98% eluent A and was maintained for 1 minute, then linearly changed to 100% eluent B within 18 minutes and maintained for 4 minutes, and finally reverted back to 98% B and equilibrated for 2 minutes. Flow rate was 0.35 mL/min, and the column temperature was kept at 55°C. The ion spray voltage was set to 4500 V. Interface heater temperature was 500°C. Curtain gas, ion source gas 1 and ion source gas 2 were set to 35 PSI, 50 PSI and 50 PSI, respectively. FFA concentrations are given in µmol/L, concentrations of metabolites analyzed by CE-TOF/MS are given in arbitrary units (AU)/L.