Agilent 6530 accurate mass qtof spectrometer

Samples were analysed using an Agilent 1260 Infinity II LC system with Agilent 6530 Accurate-Mass QToF spectrometer, using an Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HD analytical column (1.8 μm particle size, 2.1 × 50 mm) with a binary eluent system comprising MeOH / H₂O (12 min gradient: 1–99% with 0.1% formic acid) as mobile phase. Operating pressures were in the range of 2000–3000 PSI. Electrospray ionisation mass spectrometry was conducted in positive ion mode (m/z range: 50 – 3200) using a fragmentor voltage of 150 V, gas temperature of 325 °C (flow 10 L/min) and sheath gas temperature of 400 °C (flow 11 L/min). All peptides were dissolved in pure water and kept frozen (−18 °C) in aliquots prior to use.

Extracts were dissolved in MeOH at the final concentration of 0.1 mg/mL and injected onto a Phenomenex Kinetex (Torrance, CA, USA) C18 reversed-phase HPLC column (2.6 mm, 100 × 4.6 mm). Samples were analyzed using an Agilent 6530 Accurate-Mass Q-TOF spectrometer coupled to an Agilent 1260 LC system (Santa Clara, CA. USA) under the following LC conditions with 0.1 % TFA: 1–5 min (10% MeCN in H₂O), 5–26 min (10–100% MeCN), 26–30 min (100% MeCN). The divert valve was set to waste for the first 5 min. Q-TOF MS settings during the LC gradient were as follows: positive ion mode mass range 300–2500 m/z, MS scan rate 1/s, MS/MS scan rate 5/s, fixed collision energy 20 eV; source gas temperature 300°C, gas flow 11 L/min, nebulizer 45 psig, scan source parameters: VCap 3000, fragmentor 100, skimmer1 65, octopoleRFPeak 750. The MS was auto-tuned using Agilent tuning solution in positive mode before each measurement. LC (DAD) data were analyzed with ChemStation software (Agilent) and MS data were analyzed with MassHunter software (Agilent). The spectral data generated from the biological replicates were combined for each strain prior to the network analyses.