AcrB nanodiscs were equilibrated in non-deuterated AcrB sample buffer (no D 2 O labeling performed) and ice-cold nanodisc quench buffer was added 1:1 (vol/vol). Three technical replicates were performed with standard bottom-up HDX-MS workflow applying a 9.0-minute linear gradient from 8 to 40% solvent B at 40 µL/min (Figure ). The total protein amount injected was 20 pmol. Detailed LC settings are provided in Supporting Information (Table and).