Heat-treatment, Trypsin Digestion and Gel Analysis of Spike Ectodomain.
Spike ectodomain protein (Omicron variant, 1.3 μg/μL in 20 mM Tris, 300 mM NaCl, pH 8) was diluted 1:20 in HA buffer (100 mM Tris, 50 mM NaCl, pH 7.8) and 40 μL aliquots were incubated in a PCR tube and heater (Thermo Scientific) for 1 hour at 25, 35, 45, 55, and 65°C. After cooling to room temperature and centrifugation (1000 RPM, 10 min), supernatants were transferred to clean PCR tubes for trypsin digestion. Sequencing grade trypsin (Promega, WI) was diluted in HA buffer so that adding 2 μL of trypsin to each heat denatured aliquot resulted in a 1:100 molar ratio of trypsin to spike. A control set of aliquots had 2 μL of HA buffer with no trypsin added to verify heat-treatment did not result in aggregation. All tubes were incubated for 1 hour at 37°C, cooled to room temperature, measured for volume loss and compensated with HPLC-grade water. A nonreducing Bis-Tris 4-12% gradient gel (Thermo Scientific) was used to detect size-resolved spike proteins (Figure S1).