Metabolic labeling of glycans by radioactive sugars

 Prepare ~80% confluent cells cultured in 10 cm dish under specified condition.

 Wash once with glucose-free medium containing 2% dialyzed FCS.

 Replace with 4 mL of medium containing 1.85–7.4MBq (50–200μCi) of [³H]glucosamine, 1/10 concentration of glucose and 2% dialyzed FCS.

 Incubate at 37˚C for 20–24 h under 5% CO₂ atmosphere.

 Centrifuge the collected medium at 10,000 × g for 10 min, and collect the supernatant.

 Remove the radioactive components (M.W. -10kDa) with ultrafiltration device (Amicon Ultra-4), change buffer to TBS, and finally concentrate to 100–200 μL.

 Harvest the cells in PBS using a rubber cell scraper, and then centrifuge to collect cells.

 [Preparation of N-glycans]

Denature the packed cells or the solution obtained in step 6), and then digest with PNGase F according to the manufacturer's instructions at 37˚C for 18 h (See section “N-glycanase digestion”).

 Heat at 100˚C for 3 min, and then separate the released N-glycans from the reaction mixture by ultrafiltration with milli-Q water.

 Apply the filtrate as obtained above in step 9) to a Sep-Pak Vac C18 cartridge that was pre-washed with methanol and milli-Q water, and the flow-through fraction was dried using a centrifugal vacuum concentrator.

 [Preparation of O-glycans]

Transfer the residual material (0.1–0.2 mL) of the ultrafiltration device in step 9) to a 1.5 mL tube, add equal volume of 0.1 M KOH/ 2 M NaBH₄ solution, and incubate at 30˚C for 15 h.

 Transfer the reaction mixture to a 15 mL tube, and add several drops of acetic acid very carefully because a large amount of H₂ bubble is formed.

 When the bubbling ends, apply the reaction mixture to a Sep-Pac Vac C18 cartridge (3 cc), overlayered with 2 mL of AG-50W(H⁺) resin that was pre-washed with milli-Q water, and then flushed out with 10 mL of milli-Q water.

 Collect the flow-through fraction and dry it in a centrifugal vacuum concentrator.

 Add 1 mL of methanol and dry; repeat this step two more times.

 [Analysis of N-glycan and O-glycan]

Obtained N-glycans and O-glycans are subsequently available for further analysis by chromatography, such as Bio-Gel P-4 gel-permeation chromatography, Mono Q ion-exchange column chromatography, C18 reverse-phase column chromatography, Amido80 normal phase column chromatography, PGC chromatography, and serial lectin column chromatography.