Oasis max cartridge

Glycans in screw-capped glass tubes were permethylated by adding finely grounded NaOH pellets in 200 μl of dimethyl sulfoxide and 100 μl of iodomethane and incubated in a shaker at 4°C for 3 hours. The reaction was terminated by adding 20% acetic acid on ice. Excess iodomethane collected at the bottom of the tube was evaporated off by applying a gentle stream of nitrogen gas, and the neutralized reaction mixtures were then loaded onto a Waters^® OASIS MAX cartridge, which was preconditioned with 3 ml of 100% acetonitrile and 3 ml of 100 mM ammonium acetate buffer. After stepwise washing with 3 ml of ammonium acetate buffer and 9 ml of ddH₂O, the permethylated glycans were eluted successively by 6 ml of 95% acetonitrile (for neutral glycans), 6 ml of 1 mM ammonium acetate in 80% acetonitrile (single negatively charged), and 6 ml of 100 mM ammonium acetate in 60% acetonitrile and 20% methanol (double and multiple negatively charged). Each glycan fraction was further subjected to additional cleanup by C18 Ziptip^® prior to MS analysis to reduce contaminants.

The solutions after each reaction of Section 2.2, Section 2.3 and Section 2.4 were heated at 37 °C for 18 h after adding FITC/N,N-dimethylformamide (5 mg/30 µL). Acetonitrile was added to the reaction mixture, which was centrifuged (10,000× g, 10 min), and then, a solution of 0.5-M ammonium hydroxide solution was added to the supernatant. This solution was added to the Oasis MAX cartridge (Waters, Milford, MA, USA), and the cartridge was washed with acetonitrile, water, and 0.5% cyanoacetic acid/water and then eluted with 0.25% cyanoacetic acid/acetonitrile. The eluate was concentrated and dried by nitrogen purging and then dissolved in 0.1% formic acid/acetonitrile:water (1:1) to prepare the final sample.