6540 quadrupole time of flight q tof mass spectrometer

Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on a 1260 Infinity Quaternary LC System coupled with a 6540 Quadrupole Time-of-Flight (Q-TOF) mass spectrometer with Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI) ionization source (Agilent Technologies, Santa Clara, CA, USA). Samples were separated using the ZORBAX Eclipse Plus C18 Rapid Resolution High Throughput (RRHT) 2.1×100 mm 1.8 µm column (Agilent Technologies) and analyzed in both positive and negative ionization modes. For positive mode experiments, the mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). A linear gradient was run from 2% to 98% B over 25 minutes, at 0.5 µl/min. In negative mode, the mobile phases were water with 1 mM ammonium fluoride (A) and acetonitrile (B). Similarly, a linear gradient was turn from 2% to 98% B over 25 minutes, with a flow rate of 0.5 µl/min. The injection volume was 3 µL and three injections were made for each sample. ESI conditions were spray voltage 3.0 kV, gas temperature, 300°C, drying gas, 8 L/min, nebulizer 35 psig, VCap 3500 V, fragmentor 175 V and skimmer 65 V. The instrument was set to acquire over the m/z range of 100–1700 with an acquisition rate of 1 spectra/s. Three biological replicates were analyzed for each sample.