Glycopeptide Enrichment and Fractionation

The glycopeptides were enriched by mixed anion exchange (MAX) extraction cartridges and then fractionated by basic reversed-phase liquid chromatography (bRPLC) (Yang et al., 2018 (link)). In brief, 1 mg peptides were loaded into one Oasis MAX extraction cartridges (Waters) and about 200 µg glycopeptide eluate was collected together from three MAX cartridges. Then, the collected glycopeptides were desalted by C18 solid-phase extraction and injected into Zorbax Extend-C18 analytical column containing 1.8 μm particles at a flow rate of 0.2 ml/min. The glycopeptides were fractionated and collected into 96-well plates in a time-based mode from 16 to 112 min. The resulting 96 fractions of tryptic glycopeptide samples were further concentrated into 24 fractions by combining fractions 1, 25, 49, 73; 2, 26, 50, 74; etc. The 24 fraction samples were then dried in a speed-vac and stored at −80°C until LC-MS/MS analysis.