Captivespray nanobooster

IgG was enzymatically digested with trypsin and analysed by reverse phase-nanoLC-MS. Electrospray ionisation was achieved with a CaptiveSpray nanoBooster (Bruker Daltonics) using acetonitrile-enriched nitrogen gas to enhance sensitivity. Glycopeptides were detected using a quadrupole-time-of-flight (TOF) mass spectrometer (MS) (maXis impact HD ultra-high resolution QTOF; Bruker Daltonics)^{108 (link)}. Double and triple charged tryptic Fc glycopeptide signals were integrated and normalised to the subclass-specific total glycopeptide intensity. Quality of mass spectra was evaluated based on intensities of total IgG1 glycoforms. Glycosylation traits were calculated as detailed in Supplementary Table S3.

The nanoflow HPLC was coupled to the mass spectrometer with a CaptiveSpray nanoBooster ionization source (Bruker Daltonik GmbH, Bremen, Germany). The mass spectrometer was a Maxis II QTOF (Bruker Daltonik GmbH), and the data acquisition strategy was the data-dependent analysis (DDA). The spectra were collected using a fixed cycle time of 2.5 s (dynamic exclusion 2 min) and acquired at 3 Hz in the 150–2200 m/z mass range, while CID was performed at 4 or 16 Hz depending on the intensity of the precursor. The preferred charge states of precursors were mostly set from +1 to +5, but some samples were analyzed using only +1 or from +2 to +5.
The raw data were first recalibrated with Bruker Compass DataAnalysis software 4.3 (Bruker Daltonik GmbH). The peptides were identified by Byonic 4.2.10 software. The search engine parameters are detailed in Table S1.