The IgG3 (glyco)peptides were analyzed with nanoLC-reversed phase (RP)-electrospray (ESI)-ion trap (IT)-MS(/MS) on an Ultimate 3000 RSLCnano system (Dionex/Thermo Scientific) coupled to an amaZon speed ESI-IT-MS (Bruker Daltonics, Bremen, Germany). A precolumn (Acclaim PepMap C18 capillary column, 300 μm x 5 mm, particle size 5 μm, Dionex/Thermo Scientific) was used to wash and concentrate the sample, and separation was achieved on an Acclaim PepMap RSLC C18 nanocolumn (75 μm x 150 mm, particle size 2 μm, Dionex/Thermo Scientific) with a flow rate of 500 nl/min. The following linear gradient was used, with solvent A consisting of 0.1% formic acid in water and solvent B of 95% acetonitrile, 5% water: t = 0 min, 1% solvent B; t = 5 min, 1% B; t = 20 min, 25% B; t = 25 min, 70% B; t = 30 min, 70% B; t = 31 min, 1% B; t = 55 min, 1% B. The sample was ionized in positive ion mode with an ESI-nanosprayer (4500 V) using a bare fused silica capillary (internal diameter of 20 μm). The solvent was evaporated at 180 °C with a nitrogen flow of 5 liters/min. A CaptiveSpray nanoBooster (Bruker Daltonics) was mounted onto the mass spectrometer and saturated the nitrogen flow with ACN to enhance the sensitivity. The MS1 ion detection window was set at m/z 350–1400, and the MS2 window at m/z 140–2200. The three highest nonsingly charged peaks in each MS1 spectrum were automatically fragmented through collision-induced dissociation (CID). In order to identify the peptide sequence of proteinase K- and trypsin-generated O-glycopeptides, MS3 analysis was performed on manually selected precursors: the MS2 peak representing the peptide without sugars attached was targeted for fragmentation. In a separate LC-MS run, electron transfer dissociation fragmentation was done on selected precursor ions.
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