6550 ifunnel q tof lc ms mass spectrometer

Tryptic-digested peptides stored in −20 °C were reconstituted in 20 μL of 0.1% formic acid in water. 1 μL of peptide sample was injected on a nano C-18 column (see below) using the Agilent 1200 capillary nanoflow liquid chromatography system coupled to a 6550 iFunnel Q-TOF LC/MS mass spectrometer (Agilent) via a nano-electrospray (Nano-ESI) source. Digested peptide products were separated using the C18 300 Å Large Capacity Chip column (Zorbax 300SB-C18) that was pre-equilibrated with 0.1% formic acid in water (A) and eluted in a multi-gradient approach using 90% acetonitrile in 0.1% formic acid in water (B) at a flow rate of 4 μL/min. The method utilized a 47 min gradient starting with a 30 min ramping from 3% to 50% solution B, followed by a further increase of 50% to 95% solution B for 2 min, 95% solution B for 7 min, and finally a decrease from 95% to 3% solution B for another 8 min. BSA was used as a standard for reference. Mass spectrometry (MS/MS) was performed using these following settings: Gas temperature 300 °C; drying gas 5 L/min; capillary voltage of 2050 V; and fragmenter voltage 300 V. The auto MS/MS mode was used, with positive ion polarity setup. Acquisition ranges were 110–3000 m/z for MS scan and 50–3000 m/z for MS/MS scan.