Ultimate wps 3000rs nanolc system

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 proteomics and glycoproteomics samples were characterized using an UltiMate™ WPS-3000RS nanoLC system coupled with an Orbitrap Fusion Lumos (ThermoFisher Scientific). 1 μL of the sample was injected, and the analytes were separated on an Acclaim™ PepMap™ 100C18 LC Column (3 μm, 0.075 mm × 250 mm, ThermoFisher Scientific) at a flow rate of 300 nL min⁻¹. Water containing 0.1% formic acid and 80% acetonitrile containing 0.1% formic acid were used as solvents A and B, respectively. MS spectra were collected with a mass range of m/z 600–2000 at a rate of 1.5 s per spectrum in positive ionization mode. The filtered precursor ions in each MS spectrum were subjected to fragmentation through 30% higher-energy C-trap dissociation (HCD) with nitrogen gas.