Agilent masshunter data acquisition software

The digested sample was loaded into a Agilent C18 300 Å Large Capacity Chip (Agilent Technologies, Santa Clara, CA, USA). The column was equilibrated by 0.1% formic acid in MilliQ water (solution A) and peptides were eluted with an increasing gradient of 90% ACN in 0.1% formic acid (solution B) by the following gradient; 3%–50% solution B from 0–30 min, 50%–95% solution B from 30–32 min, 95% solution B from 32–39 min and 95%–3% solution B from 39–47 min. The polarity of the Q-TOF was set at positive, capillary voltage at 2150 V, fragmentor voltage at 300 V drying gas flow of 5 L/min and gas temperature of 300 °C. The spectrum was scanned in auto MS/MS mode over a range of 110–3000 m/z for MS scan and 50–3000 m/z range for MS/MS scan. The spectrum was analyzed by using Agilent MassHunter data acquisition software (Agilent Technologies, Santa Clara, CA, USA).

Digested peptides from in-solution tryptic digestion were loaded into an Agilent C18 300 Å Large Capacity Chip (Agilent Technologies, Santa Clara, CA, USA) and equilibrated with 0.1% formic acid in water (solution A). The peptides were then eluted from the column with an increasing gradient of 90% acetonitrile in 0.1% formic acid in water (solution B) by the following gradient; 3–50% solution B from 0–30 min, 50–95% solution B from 30–32 min, 95% solution B from 32–39 min and 95–3% solution B from 39–47 min. Q-TOF polarity was set at positive with capillary and fragmenter voltage being set at 2050 V and 300 V, respectively, and 5 L/min of gas flow with a temperature of 300 °C. The intact protein spectrum was analyzed in auto MS mode ranging from 110–3000 m/z for MS scan and 50–3000 m/z for MS/MS scan. The spectrum was then analyzed with Agilent MassHunter data acquisition software (version B.07.00, Agilent Technologies, Santa Clara, CA, USA) and then PEAKS 7.0 software (Bioinformatics Solutions Inc., Waterloo, ON, Canada).

10 μL of the prepared sample containing phospholipid was injected into an Agilent 1200 HPLC system (Agilent). 50% (v/v) of water with 0.1% (v/v) formic acid and 50% (v/v) acetonitrile with 0.1% (v/v) formic acid was used as an isocratic mobile phase at a flow rate of 0.4 mL/min. The HPLC system was connected to an Agilent 6220 Accurate-Mass TOF (Agilent) equipped with an electrospray interface operating in positive ion mode. The optimized operation parameters were as follows: capillary voltage, 4000 V; nebulizer pressure, 30 psi; drying gas flow rate, 11 L/min; gas temperature, 673 K; skimmer voltage, 60 V; octapole rf, 250 V; fragment voltage (in-source CID fragmentation), 200 V. LC-MS accurate mass spectra were recorded across the range m/z 50–1000. The accurate-mass calibration was performed over a mass range of m/z 118.0863–2721.8950 using a calibration solution provided by the manufacturer (G1969-85000, Agilent, Santa Clara). The full-scan data were recorded with the Agilent Mass Hunter Data Acquisition software (version B.03.01) and processed with the Agilent Mass Hunter Qualitative Analysis software (version B.03.01).