Paramagnetic Bead-Based Protein Cleanup and Glycopeptide Enrichment

The
paramagnetic beads, i.e., Sera-Mag SpeedBead Carboxylate-Modified
[E3] Magnetic Particles (Cytiva) and Sera-Mag SpeedBead Carboxylate-Modified
[E7] Magnetic Particles (Cytiva), were used for sample cleanup. The
beads were washed with water twice before use. The beads were added
to the lysate in a ratio of 10:1 (wt/wt, beads to proteins) based
on the well-established protocol.^{15 (link)} In
the bead–lysate mixture, acetonitrile (ACN) was slowly added
with constant swirling to ensure that the beads did not stick to the
conical tube wall until ACN reached a final concentration of 95%.
The mixture was incubated at 37 °C for 20 min to maximize the
binding of peptides and proteins to the beads. Then the tubes were
placed in magnetic racks, and the unbound supernatant was removed
after the beads migrated to the tube wall. The beads were washed with
95% ACN three times. A digestion buffer containing 50 mM HEPES, pH
= 8.6, 1.6 M urea, and sequence grade trypsin (Promega) was added,
followed by incubation for 16 h at 37 °C to digest proteins.
The peptide solutions were acidified and desalted using the tC18 Sep-Pak
cartridges (Waters). Glycopeptides were enriched using NeutrAvidin
resins in PBS at room temperature for 1 h. The resins were washed
with PBS for eight times and water for two times to remove nonspecific
binding peptides. The enriched glycopeptides were eluted twice under
UV radiation, each for 1 h at room temperature.