Glycomic Sample Preparation and N-Glycan Isolation

Details of the glycomic sample preparation have been described previously ((Wu et al., 2010 (link); Wu et al., 2011 (link))). Extracted cell membrane fractions or protein (RNase B) were suspended with 100 μl of 100 mM NH₄HCO₃ in 5 mM dithiothreitol and heated in boiling water for 2 min to denature the proteins. Solutions of with 2 μl of peptide N-glycosidase F (New England Biolabs, MA, United States) were added to the samples, and the resulting solutions were then incubated in a microwave reactor (CEM Corporation, NC, United States) at 20 W, 37°C for 10 min. The samples were further placed in a 37°C water bath for 18 h. Ultracentrifugation at 200,000 × g for 45 min was performed to precipitate proteins, and the supernatant containing N-glycans was collected and desalted using porous graphitic carbon (PGC) on a 96-well SPE plate (Grace, IL, United States). The plate was equilibrated with 80% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Then the samples were loaded onto the plate and washed with nanopure water. N-Glycans were eluted with a solution of 40% (v/v) acetonitrile containing 0.05% (v/v) trifluoroacetic acid, and dried in vacuo using miVac (SP Scientific, PA, United States) prior to further analysis.