The IgG glycopeptide samples were analysed using liquid chromatography coupled to mass spectrometry (LC-MS), in a setup described previously41 (link). An amount of 2.5 µL of the samples was injected in an Ultimate 3000 RSLCnano liquid chromatography system (Dionex, Sunnyvale, CA). The samples were first washed on an Acclaim PepMap100 C18 trap column (5 mm × 300 µm i.d., Dionex, Sunnyvale, CA), and subsequently separated on an Ascentis Express C18 nanoLC column (50 mm × 75 µm i.d., 2.7 µm HALO fused core particles; Supelco, Bellefonte, PA) with a flow rate of 0.9 µL/min. The following linear gradient was used, with solvent A consisting of 0.1% trifluoroacetic acid and B of 95% acetonitrile (ACN): t = 0, 3% solvent B; t = 2, 6%; t = 4.5, 18%; t = 5, 30%; t = 7, 30%; t = 8, 0%; t = 11, 0%.
Via a sheath-flow electrospray (ESI) interface (Agilent Technologies, Santa Clara, CA), the LC was coupled to a Maxis Impact quadrupole time-of-flight (QTOF)-MS system (micrOTOF-Q; Bruker Daltonics, Bremen, Germany). A sheath-flow consisting of 50% isopropanol, 20% propionic acid (Merck) and 30% MilliQ-purified water was applied at 2 µL/min, and nitrogen gas was applied at 4 L/min. MS1 spectra were acquired with a frequency of 0.5 Hz and within an m/z range of 600-2000. An IgG standard and two blank injections were run in between every 12 runs.
Via a sheath-flow electrospray (ESI) interface (Agilent Technologies, Santa Clara, CA), the LC was coupled to a Maxis Impact quadrupole time-of-flight (QTOF)-MS system (micrOTOF-Q; Bruker Daltonics, Bremen, Germany). A sheath-flow consisting of 50% isopropanol, 20% propionic acid (Merck) and 30% MilliQ-purified water was applied at 2 µL/min, and nitrogen gas was applied at 4 L/min. MS1 spectra were acquired with a frequency of 0.5 Hz and within an m/z range of 600-2000. An IgG standard and two blank injections were run in between every 12 runs.