Agilent 6520 accurate mass q tof esi mass spectrometer

Purified proteins (3–12 μM) were incubated with relevant cofactors. Reactions (25–50 μL) were extracted with ethyl acetate, with the organic phase dried and reconstituted in 88 μL of methanol for analysis by LC-MS (Agilent 1260 HPLC with Agilent 6520 Accurate-Mass Q-TOF ESI mass spectrometer).

Samples were analyzed by reversed-phase chromatography on an Agilent 1260 HPLC, using a 5 μm, 2 × 100 mm Gemini NX-C₁₈ column (Phenomenex). Water with 0.1% formic acid (A) and Acetonitirile with 0.1% formic acid (B) were used as the mobile phase components at a flow rate of 0.4 mL/min with the following 55 min gradient; i) for methanol extracts: 0–30 min, 3–50% B; 30–45 min, 50–97% B; 45–50 min, 97% B; 50–51 min, 97–3% B; 51–55 min, 3% B, ii) for saponified extracts: 0–45 min, 40–97% B; 45–50 min, 97% B; 50–51 min, 97–40% B; 51–55 min, 40% B. A coupled Agilent 6520 Accurate-Mass Q-TOF ESI mass spectrometer was used to collect MS data in positive ion mode (parameters: mass range: 100–1700 m/z; drying gas: 300°C, 1 L/min; nebulizer: 25 psig; capillary: 3500 V; fragmentor: 150 V; skimmer: 65 V; OCT 1 RF Vpp: 750 V; 1000 ms per spectrum). The first minute of each run was discarded to avoid salt contamination of the MS apparatus. For tandem mass spectrometry (MS/MS) analysis, 5, 10, 20 and 40 V collision energies were used with an m/z window of 1.3 centered on the m/z analyzed.