3.5. N-Glycan Release, Labeling and Clean-Up

The glycan release was performed using 9 µL of serum sample, according to the PNGase F deglycosylation protocol of New England Biolabs (Ipswich, MA, USA). The released glycans were labeled by the addition of 10 μL 0.3 M procainamide and 300 mM picoline borane in 70%/30% dimethyl sulfoxide/acetic acid and incubated for 4 h at 65 °C. After the PNGase F digestion and fluorescent labeling, the sample was suspended with the magnetic nanoparticles in 90% acetonitrile. As these particles are hydrophilic, they create hydrogen bonds with water molecules; however, when the water concentration is decreased (90% acetonitrile, 10% water), they connect to other hydrophilic molecules such as sugars. At the end of the purification, the carbohydrates can be eluted from the magnetic nanoparticles by water and analyzed by LC.