Compass data analysis version 4.2

Electrochemical oxidation was performed using a ROXY Potentiostat (Antec Scientific, Zoeterwoude, The Netherlands) equipped with a µPrepCell 2.0 containing a Magic Diamond™ working electrode (boron doped diamond). The oxidation was achieved by applying a ramping voltage (2.8 to 3.2 V—in steps of 20 mV/s) at 37 °C and the products obtained were analyzed by on-line coupling of the electrochemical cell with an ESI-HCT Ion Trap MS (Bruker, Bremen, Germany); 25 µmol/L of SM(d18:1/18:1) standard in MeOH:20 mM ammonium formate (1:1 v/v) was infused at the flow rate of 5 µL/min to the ROXY EC cell directly coupled to the ESI-MS. The electrospray voltage was set at 4.2 kV; the capillary temperature at 300 °C; sheath gas pressure was 20.00 psi; drying gas flow rate was 5.00 standard liter per minute. The analysis was done in positive mode, and the most intense ions were selected for MS/MS analysis from each full-scan mass spectrum, followed by the dynamic exclusion for 20 min. MS/MS spectra were acquired in profile mode; isolation width was 4.00 m/z units, and fragmentation time was 40 ms. Data were analyzed using Compass Data Analysis (version 4.2) and Compass Data Analysis Viewer (version 4.2) (Bruker, Billerica, MA, USA).

Mass spectrometry N- and O-glycan spectra were evaluated manually by Compass DataAnalysis version 4.2 (Bruker). The data were then loaded into QuantAnalysis version 2.2 (Bruker) where quantitation was performed on extracted ion chromatograms (XIC) including all detected charge states of the respective glycan. A smoothing width of 3 s (Gauss algorithm) was applied on each XIC.
Data from xCGE-LIF aquisition data was analyzed using the Java-based glycan analysis software glyXtool™ (glyXera, Magdeburg, Germany). Data were normalized to an internal standard, transforming electropherograms to “N-glycan fingerprints” (see Figure S9 in Supplementary Material). Automated peak picking, integration, and relative quantification were performed, followed by N-glycan assignment to peaks via migration time matching to an in-house N-glycan database. N-glycan sequences and linkages were confirmed by exoglycosidase digests (see Supplementary Material).

LC-MS analysis was performed on an Agilent 1260 series HPLC system equipped with a Multospher 120 RP-18 column (250 × 2 mm, 5 μm, CS-Chromatographie Service, Langerwehe, Germany). Solvents A (water) and B (acetonitrile), each substituted with 0.1% formic acid, were used as mobile phases with a flow rate of 0.5 mL min⁻¹ with a linear gradient of 5 to 100% B in 10 min for analysis of the in vitro assays. For analysis of the samples of the biotransformation experiments, a flow rate of 0.25 mL min⁻¹ with a linear gradient of 5 to 100% B in 40 min was used. Positive ion mode electrospray ionization (ESI) in a micrOTOF-Q III mass spectrometer by Bruker Daltonics (Bremen, Germany) was used for mass detection. The parameters were set as follows: capillary voltage of 4.5 kV and a collision energy of 8.0 eV. LC-MS data were processed using Bruker Compass Data Analysis version 4.2 (Build 383.1) software.