Enzymatic release and solid-phase extraction of N-glycans were performed according to optimized procedures published by Kronewitter et al.26 (link) Briefly, membrane glycoproteins were denatured by rapid thermal cycling (25–100 °C) in an aqueous solution of 100 mM ammonium bicarbonate and 5 mM dithiothreitol. Next, 2.0 μL (or 1000 U) of peptide N-glycosidase F (New England Biolabs) were added and the mixture was incubated in a microwave reactor (CEM Corporation) for 10 minutes at 20 watts. Following the addition of 800 μL of cold ethanol, the mixture was chilled at −80 °C for 1 hour, then centrifuged in order to precipitate out the deglycosylated proteins. The glycan-rich supernatant fraction was collected and dried in vacuo.
Graphitized carbon solid-phase extraction was performed using an automated liquid handler (Gilson). Graphitized carbon cartridges (150 mg, 4.0 mL, Grace Davison) were washed with 80% acetonitrile / 0.10% trifluoroacetic acid (v/v) in water, then conditioned with pure water. Aqueous N-glycan solutions were loaded onto the cartridges and washed with pure water to remove salts and buffer. Membrane N-glycans were eluted with sequential addition of 10% acetonitrile, 20% acetonitrile, and 40% acetonitrile / 0.05% trifluoroacetic acid (v/v) in water. Samples were dried in vacuo.
Graphitized carbon solid-phase extraction was performed using an automated liquid handler (Gilson). Graphitized carbon cartridges (150 mg, 4.0 mL, Grace Davison) were washed with 80% acetonitrile / 0.10% trifluoroacetic acid (v/v) in water, then conditioned with pure water. Aqueous N-glycan solutions were loaded onto the cartridges and washed with pure water to remove salts and buffer. Membrane N-glycans were eluted with sequential addition of 10% acetonitrile, 20% acetonitrile, and 40% acetonitrile / 0.05% trifluoroacetic acid (v/v) in water. Samples were dried in vacuo.
