Alpha lytic protease

Nine μg of CoV-HKU1 HE were incubated in 100 mM Tris pH 8.5, 2% sodium deoxycholate, 10 mM tris(2-carboxyethyl)phosphine, and 40 mM iodoacetamide at 95 °C for ten minutes and at 25 °C for 30 min in the dark. Denatured, reduced and alkylated CoV-HKU1 HE (3 μg) was then diluted into fresh 50 mM ammonium bicarbonate and incubated overnight at 37 °C either with 0.056 μg of trypsin (Promega), chymotrypsin (Sigma Aldrich) or alpha-lytic protease (Sigma Aldrich). Formic acid was then added to a final concentration of 2% and the samples were centrifuged at 21,100 × g for 20 min at 4 °C, followed by another round of centrifugation for 5 min to precipitate the sodium deoxycholate and collect the peptides from the supernatants. Then, the CoV-HKU1 HE tryptic, chymotryptic and alpha lytic protease digests were desalted using 30 µm Oasis HLB 96-well plate (Waters). The Oasis HLB sorbent was activated with 100% acetonitrile and subsequently equilibrated with 10% formic acid in water. Next, peptides were bound to the sorbent, washed twice with 10% formic acid in water and eluted with 100 µL of 50% acetonitrile/5%formic acid (v/v). The eluted peptides were vacuum-dried and resuspended in 100 µL of 2% formic acid in water.