Extraction and Enrichment of N-Glycans

N-glycans were extracted from slides as described previously and dried by vacuum centrifugation [20 (link)]. The ethyl esterification protocol, including the modification and enrichment, was adapted from Reiding etal. [22 (link)]. Briefly, 2 µL water and 40 µL 0.25 M HOBt/EDC were added to dried glycans followed by incubation at 37 °C for 1 h. 40 µL acetonitrile was added and the mixture was placed at −20 °C for 20 min. Glycans were enriched using cotton-HILIC tips according to Selman etal. [26 (link)]. Briefly, cotton wool composed of 100% cotton (Assured, Rio Rancho, NM, USA) was inserted in 20 µL tips and equilibrated with 10 µL water three times followed by 10 µL 85% ACN three times. Samples were loaded and unbound material removed by washing three times with 10 µL 85% acetonitrile with and without 1% TFA, respectively. Tip-bound glycans were eluted in 10 µL water. Enriched and modified glycans were spotted on an Anchorchip MALDI plate (Bruker Daltonics) with 2,5-dihydroxybenzoic acid (DHB) at a concentration of 5 mg/mL in 50% ACN/50% water/1 mM NaOH. Ethanol was then used for recrystallization.