Enrichment and Analysis of N-Glycopeptides

As previously described, the SPEG method was used to capture N-linked glycopeptides (16 (link)). C18 column (Waters, Sep-Pak Vac) was used for peptide desalting, and tryptic peptides were eluted using 80% of acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA), oxidized with sodium periodate at a final concentration of 10 mM, and incubated for 1 h in the dark at room temperature. The oxidation reaction was quenched using sodium sulfite at a final concentration of 60 mM for 10 min at room temperature. The samples were coupled to hydrazide resin (Bio-Rad, Hercules, CA) in 80% ACN and 0.1% TFA for 4 h at room temperature. Then the resin was washing three times by using 100 mM NH₄HCO₃, 1.5 M NaCl, and H₂O to remove non-glycosylated peptides. N-linked glycopeptides were released from the resin by mixing with 1 μl PNGase F (New England Biolabs, Ipswich, MA) in 100 mM NH₄HCO₃ at 37°C overnight. After the C18 cartridge, the purified glycopeptides were dried by Speed-Vac and resuspended with 0.4% acetic acid for further use of LC-MS/MS analysis.