Pgc nano chip

The detailed procedures were reported previously.^{15 (link)} Briefly, the N-glycans were released from cell membrane using PNGase F after overnight incubation at 37 °C. The released N-glycans were purified using porous graphitic carbon (PGC) SPE plates, the glycan samples were analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC nano-chip (Agilent, CA), and the results were extracted with the MassHunter Qualitative Analysis B08 software (Agilent, CA). For glycoproteomic analysis, glycopeptides after trypsin digestion were enriched by solid-phase extraction using iSPE®-HILIC cartridges (Nest Group, MA). The enriched glycopeptides were characterized using a UltiMate™ WPS-3000RS nanoLC system coupled with an Orbitrap Fusion Lumos (ThermoFisher Scientific), and an in-house human N-glycan database was applied to the raw results using the Byonic software (Protein Metrics, CA).

The cell membrane fractions were resuspended with 100 μL of 5 mM DTT in 100 mM ammonium bicarbonate. The mixture was heated in boiling water for 3 minutes. The cleavage of N-glycans was performed by adding 2 μL of PNGase F followed by incubation in a 37 °C water bath overnight. The released N-glycans were separated using centrifugation at 200 000×g for 30 minutes, and the supernatant was purified using porous graphitic carbon (PGC) on an SPE plate. The glycan samples were dried and reconstituted in 30 μL nanopure water. 5 μL of the sample was injected and analyzed with an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC nano-chip (Agilent, CA). A binary gradient using solvent A with 3% (v/v) ACN and 0.1% (v/v) formic acid in water and solvent B with 90% (v/v) ACN and 1% (v/v) formic acid in water was applied to separate N-glycans at a flow rate of 300 nL min⁻¹. The resulting chromatographs of glycans were extracted with the MassHunter Qualitative Analysis B08 software (Agilent, CA). N-glycan compounds were identified with an in-house library that contains the accurate mass and formula of human N-glycans, and the N-glycan structures were confirmed through tandem MS fragmentation.

Details of the glycomic MS analysis have been described previously ((Wu et al., 2010 (link); Wu et al., 2011 (link))). Glycan samples were reconstituted with 30 μl nanopure water and analyzed using an Agilent 6520 Accurate Mass Q-TOF LC/MS equipped with a PGC nano-chip (Agilent Technologies, CA, United States). The glycan separation was performed at a constant flow rate of 300 nl min −1, and a binary gradient was applied using (A) 0.1% (v/v) formic acid in 3% acetonitrile and (B) 1% (v/v) formic acid in 90% acetonitrile: 0–2 min, 0–0% (B); 2–20 min, 0–16% (B); 20–40 min, 16%–72% (B); 40–42 min, 72–100% (B); 42–52 min, 100–100% (B); 52–54 min, 100–0% (B); 54–65 min, 0–0% (B). MS spectra within the mass range of m/z 600–2000 were collected at a rate of 1.5 s per spectrum in positive ionization mode. The most abundant precursor ions in each MS1 spectrum were subjected to fragmentation through collision-induced dissociation (CID) based on the equation V collision = 1.8 × (m/z)/100–2.4 V.