Phoenix st

Intact glycopeptides were analyzed by Orbitrap Fusion Lumos Tribrid^™ (Thermo Scientific) combined with Easy nLC 1200 UPLC system (Thermo Scientific). Samples were reconstituted with 3 % ACN in 0.1 % formic acid (solvent A) and loaded onto an in-house packed column (0.75 μm I.D. × 27.5 cm length packed with ReproSil-Pur 120 C18-AQ, 1.9 μm). Loaded peptides were subjected to the gradient with 200 μL/min of flow rate as follows: 2 to 6 % B (90 % ACN 0.1 % F.A) for 1 min, 6 to 30 % B for 84 min, 30 to 60 % B for 9 min, 60 to 90 % B for 1min, isocratic 90 % B for 5 min, 90 to 50 % B for 1 min, and isocratic 50 % B for 9 min. The temperature of the column was maintained by a column heater (Phoenix-ST) at 50°C. The ion source of the mass spectrometer was set up with 1.8 kV of electrospray voltage and 305°C of ion transfer tube temperature. The precursor ion scan was acquired with 60 K resolution at 200 m/z for from 500 to 2000 m/z range and AGC value was set as 5×10⁵. Precursor ions isolated from 0.7 m/z width were fragmented by HCD with 35 % NCE and fragment ions were acquired with 50 K resolution with 1×10⁵ of AGC value for 100 ms of injection time for a total duty cycle (2 sec). The peptide charge state screening was enabled to include 2 to 6+ ions with a dynamic exclusion time of 45 sec to discriminate against previously analyzed ions between +/− 10 ppm.