Hdx nanoacquity system

Free hexon or hexon-pVIn (at a 1:4 molar ratio) was prepared at a concentration of 10 µM in 150 mM ammonium acetate pH 5. At the start of the exchange reaction, the sample was diluted six-fold into D₂O to a final volume of 30 µL. Exposure was carried out for 10 seconds, 1, 60 and 120 minutes in triplicate at 25°C. The reaction was quenched by 1:1 mixing with ice-cold 4 M urea, 200 mM TCEP and adjusted with HCl to give a final pH upon mixing of 2.5. Immediately after quenching, the sample was injected into a Waters HDX/ nanoAcquity system for digestion on an online pepsin column (25 °C, at a flow-rate of 50 µL.min⁻¹) followed by separation on a 10 minute RP-UPLC gradient at 0 °C and MS on a Waters Xevo QToF G2. For peptide identification, hexon was prepared under identical conditions in H₂O and analyzed using MS_e data acquisition. Data for peptide identification was processed with ProteinLynx Global Server 2.5 software. Deuterium uptake was calculated compared to the control samples in H₂O using Waters DynamX 1.0.0 software. Back-exchange was estimated at approximately 30% in our workflow. No corrections for back-exchange were applied since the analysis focuses on relative changes in deuterium uptake, rather than on absolute levels. Observed changes in deuterium uptake were filtered to p<0.05 in an upaired two-tailed Student’s t-test between free and bound hexon.