NanoHPLC Chip-Q-TOF MS analysis

All samples were analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a microfluidic chip, which incorporates an enrichment column, an analytical column, and a nanoelectrospray tip in a single assembly. Purified N-glycan and O-glycan samples were separately reconstituted in 30 μL water and analyzed with a PGC chip. The binary gradient consisted of (A1) 0.1% formic acid and 3% acetonitrile in water and (B1) 1% formic acid and 89% acetonitrile in water. The LC was programmed to hold the solvent composition at 100% A1 from 0 to 2.5 min, linearly ramp to 16% B1 at 20 min and further increase B1 to 58% at 35 min at a constant flow rate of 0.3 μL/min. For isotopologue data acquisition, the Q-TOF MS was set to acquire only MS¹ in positive ionization mode with a cycle time of 1.5 s. To assist with compound identification, control samples were also run with collision-induced dissociation (CID) using nitrogen gas. Four MS² spectra were obtained for every MS¹ through data-dependent acquisition, with a total cycle time of 5.25 s. The mass range was set at 600–2000 m/z for N-glycan analysis and at 300–2000 m/z for O-glycan analysis. The instrument was calibrated with the ESI tuning mix commercially available from Agilent.

Purified intact GSL samples were reconstituted in 50 μL 1:1 methanol/water and analyzed with a C18 microfluidic chip. A binary gradient consisting of (A2) 20 mM ammonium acetate and 0.1% acetic acid in water, and (B2) 20 mM ammonium acetate and 0.1% acetic acid in 85:15 (v/v) methanol/isopropanol was used to separate the GSLs at a flow rate of 0.3 μL/min. The acquisition method was programmed to increase the percentage of B2 from 70 to 85% over 4 min, then to 100% at 40 min. The Q-TOF MS was programmed with the same parameters as the N-glycan analysis settings, described in the preceding paragraph. The mass range was set at 600–2000 m/z for GSL analysis. In all acquisition methods, the columns were flushed with 100% of solvent B for 10 min and equilibrated with the initial solvent composition for another 10 min prior to sample injection.