The extended HDX valve configuration was used and equipped with a ZrO 2 -packed phospholipid trap column (kept on ice) upstream an in-house packed pepsin column (kept at 15 C). The HDX manager was equipped with a Vanguard column (BEH C18, 130 Å, 1.7 m, 2.1 mm x 5 mm; Waters) and an Acquity UPLC column (BEH C18, 130 Å, 1.7 m, 1.0 mm x 100 mm; Waters) for peptide trapping and separation, respectively. Deuterium labeling was performed with a PAL3 RTC HDX robot (Trajan Scientific, Morrisville, US). MSP1E3D1 nanodiscs, both POPC (1:85 protein:lipid) and EPL (1:60 protein:lipid), were diluted 20-fold (95% D 2 O final) into deuterated labeling buffer (20 mM Tris, 100 mM NaCl, 0.5 mM EDTA, pH read 7.0) for 10, 100, 1,000, and 10,000 seconds at 20 C. References were performed in nondeuterated equilibration buffer. The reaction was quenched by adding 1:1 (vol/vol) ice-cold nanodisc quench buffer (500 mM glycine-HCl, 1.6 M guanidinium-HCl, 0.8 mM Na-cholate, pH 2.35). Three technical replicates were performed with standard bottom-up HDX-MS workflow applying a 7.5-minute linear gradient from 8 to 35% solvent B at 40 L/min. Peptides were measured in positive ion mode between 50 and 2,000 m/z on the Xevo G2-XS mass spectrometer, applying settings to minimize gas-phase back-exchange . The phospholipid trap column was cleaned with 3% NH 4 OH in methanol and re-equilibrated in solvent A during the subsequent wash run. Labeling experiments were also performed on free soluble MSP1E3D1 (95% D 2 O for 10, 100, 1,000, and 10,000 sec), which was measured on both standard two-and extended three-valve configuration. Detailed LC settings are provided in Supporting Information (Table and).