Mass Spectrometry

Mass spectrometry experiments on selected 2D gel spots were performed by the Proteomics platform of the CHU de Quebec Research Center, Quebec, Canada. Samples were analyzed by nanoLC/MSMS using a Dionex UltiMate 3000 nanoRSLC chromatography system (Thermo Fisher Scientific, San Jose, CA, USA) connected to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Peptides were trapped at 20 µL/min in loading solvent (2% acetonitrile, 0.05% TFA) on a 5 mm × 300 µm C18 pepmap cartridge pre-column (Thermo Fisher Scientific/Dionex Softron GmbH, Germering, Germany) over 5 min. The pre-column was then switched online with a Pepmap Acclaim column (Thermo Fisher Scientific, San Jose, CA, USA)—a 50 cm × 75 µm internal diameter separation column—and the peptides were eluted with a linear gradient from 5–40% solvent B (A: 0.1% formic acid, B: 80% acetonitrile, 0.1% formic acid) over 35 min, at 300 nL/min. Mass spectra were acquired using a data dependent acquisition mode with Thermo XCalibur software version 4.1.50. Full scan mass spectra (350 to 1800 *m*/*z*) were acquired in the orbitrap using an AGC target of 4e5, a maximum injection time of 50 ms and a resolution of 120,000. Internal calibration was done using lock mass on the *m*/*z* 445.12003 siloxane ion. Each MS scan was followed by MSMS fragmentation of the most intense ions for a total cycle time of 3 s (top speed mode). The selected ions were isolated using the quadrupole analyzer in a window of 1.6 *m*/*z* and fragmented by Higher energy Collision-induced Dissociation (HCD) with 35% of collision energy. The resulting fragments were detected by the linear ion trap at rapid scan rate with an AGC target of 1e4 and a maximum injection time of 50 ms. Dynamic exclusion of previously fragmented peptides was set for a period of 20 s and a tolerance of 10 ppm.
