Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2

Asif Shajahan; Nitin T. Supekar; Anne S. Gleinich; Parastoo Azadi

The protein subunits S1 and S2 as HEK 293 culture supernatants were fractionated on separate lanes using SDS-PAGE. The gel was stained by Coomassie dye and the bands corresponding to subunit 1 (200 to 100 kDa) and subunit 2 (150 to 80 kDa) were cut into smaller pieces (1 mm squares approx.) and transferred to clean tubes. The gel pieces were de-stained by adding 100 µL acetonitrile (ACN): 50mM NH4HCO3 (1:1) and incubated at room temperature (RT) for about 30 min. Tubes were centrifuged, the supernatant was discarded, and 100 µL ACN was added before incubation for 30 min. The proteins on gel pieces were reduced by adding 350 µL 25 mM DTT and incubating at 60 °C for 30 min. The tubes were cooled to RT and the supernatant removed. The gels were washed with 500 µL of ACN, 350 µL 90 mM IAA was added, and the mixture was incubated at RT for 20 min in the dark. Proteins were digested by adding sequence-grade trypsin and/or chymotrypsin (we performed digests with both enzymes individually and as a cocktail) in digestion buffer (50 mM NH4HCO3) for 18 h at 37 °C separately.