Quantitative Analysis of O-Glycans via PGC-LC-MS/MS

The O-glycan samples were then reconstituted in 20 μl of MQ water, and 2 μl were injected for analysis. Analysis was performed using a PGC nano-LC Ultimate 3000 UHPLC system (Thermo Fisher Scientific) coupled to an amaZon ETD speed ion trap (Bruker Daltonics). The samples were loaded using 100% buffer A (10 mM ABC) at a loading flow of 6 μl/min on a custom-made trap column (size 30 × 0.32 mm) packed with 5 μm particle size PGC stationary phase from Hypercarb PGC analytical column (size 100 × 4.6 mm, 5 μm particle size; Thermo Fisher Scientific). Afterward, the O-glycans were separated at a 0.6 μl/min flow rate on a custom-made PGC column (100 × 0.1 mm, 3 μm particle size obtained from Thermo Fisher Scientific) by applying a linear gradient from 1% to 50% buffer B (MeCN, 10 mM ABC) over 73 min. During the procedures, a constant column temperature of 45 °C was maintained. To continue, the LC system was coupled to an amaZon ETD speed electrospray ionization (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) flow rate was set to 3 l/min, and the temperature was set at 280 ˚C. The nebulizer gas pressure was kept at 3 psi. The nanoBooster bottle (Bruker Daltonics) was filled with methanol, as a dopant solvent (34 (link)). MS spectra were acquired in enhanced mode within a mass to charge ratio (m/z) range of 380 to 1850. The maximum acquisition time was set to 200 ms, the ion charge control (ICC) to 40,000, and the target mass of smart parameter setting was set to m/z 900. MS/MS spectra were generated by collision-induced dissociation of the three most abundant precursors, applying an isolation width of 3 Thomson. In addition, ICC was set to 150,000, and the fragmentation cutoff was set to 27% with a 100% fragmentation amplitude using the Enhanced SmartFrag option (30–120% in 32 ms). To integrate area under the curve for each individual glycan isomer, extracted ion chromatograms of the first three isotopes were used in Bruker Compass DataAnalysis software (version 5.0). Peaks were manually picked and integrated. Total area normalization was employed for relative quantification of O-glycan species. Identification of O-glycan species was performed by comparison with PGC retention time, MS/MS spectra, and the BSM standard.