PGC-LC-MS/MS measurements

Prior to analysis, the samples were resuspended in 15 µL of MQ and 1 µL of each sample was pooled together in a separate vial for a quality check and method optimization (QC). The QC pool and a bovine submaxillary mucin standard were used to check instrument performance each day of measurement. A total of 6 µL of each tissue sample was injected for analysis (40%). A custom-made trap column (size 30 × 0.32 mm) packed with 5 μm particle size PGC stationary phase was used to load the samples using 100% buffer A (10 mM ammonium bicarbonate) at a loading flow of 6 μL/min. The packing material was obtained from Hypercarb PGC analytical column (size 100 × 4.6 mm, 5 μm particle size, Thermo Fisher Scientific, Waltham, MA). The glycans were separated on a custom-made PGC column (100 mm × 75 μm, 3 μm particle size; packing material obtained from Thermo Fisher Scientific, Waltham, MA) at a 0.6 μL/min flow rate by applying a linear gradient from 1% to 50% of buffer B (60% acetonitrile, 10 mM ammonium bicarbonate) over 73 min. A constant column temperature of 45 ˚C was maintained. A part of the samples were remeasured to resolve isomers with a composition of H2N2F1S1 (isomer c and f). A different gradient was used for this purpose, ranging from 1% to 50% of buffer B over 110 minutes at 35 ˚C. The peak ratios of the resolved peaks were taken to extrapolate the peak areas from the original measurement (Table S11). The LC system was coupled to an amaZon ETD speed ESI ion trap MS using the CaptiveSpray™ source (Bruker Daltonics) with an applied capillary voltage of 1000 V in negative-ionization mode. The drying gas (N₂) temperature was set at 280˚C and the flow to 3 L/min. The nebulizer gas pressure was kept at 3 psi enriched with isopropanol as described before ²². MS spectra were acquired in enhanced mode within a mass to charge ratio (m/z) range of 380-1850, target mass of smart parameter setting was set to m/z 900, ion charge control (ICC) to 40,000 and maximum acquisition time to 200 ms. MS/MS spectra were generated by collision-induced dissociation on the three most abundant precursors, applying an isolation width of 3 Th. The fragmentation cut-off was set to 27% with 100% fragmentation amplitude using the Enhanced SmartFrag option (30-120% in 32 ms) and ICC was set to 150,000.