Furthermore, no differences in the chromatographic profile are present across all chromatograms, indicating that the blocking reagent does not get released over time. For peptide identification we not only targeted MSP1E3D1, but also the blocking protein BSA and the primary digestion reagent pepsin. Free MSP1E3D1 yielded a total number of 122 identified peptides in the two-valve configuration compared to 116 peptides obtained for MSP1E3D1 in POPC nanodiscs measured with the three-valve system (Figure ). Of interest, the amount of non-MSP1E3D1 peptides account for approximately 10% in both experiments, confirming that the phospholipid trap column is only releasing small quantities of the blocking protein. Full sequence coverage could be obtained for MSP1E3D1 in nanodiscs, measured with the threevalve system (Figure ) -even slightly better than for free MSP1E3D1 (Figure ) -demonstrating the functionality of the automated HDX-MS workflow with online delipidation of nanodisc samples. Finally, we performed deuterium labeling experiments (ranging from 10 to 10,000 sec) on POPC and EPL nanodiscs as well as on free MSP1E3D1. The deuteration of backbone amide hydrogens in native proteins is mediated by transient opening/closing events in their H-bonding networks . We observed that MSP1E3D1 predominantly displays structural dynamics following EX1 kinetic regimes, both in its lipid-free soluble form and when it encases lipids to form nanodiscs (Figure ). To note, we were able to verify that the isotopic envelope bimodality observed across several peptides of the free MSP1E3D1 does not result from artefacts, e.g peptide carry-over, induced by the three-valve system, as it was observed, and at comparable extent, also for the free form analyzed with the standard two-valve system (Figure ). Importantly, and in line with previous reports , we found that nanodiscs formation leads to stabilization of MSP1E3D1, which is displayed by more intense low-mass envelopes in the EX1 regime of MSP1E3D1 peptides in both nanodiscs forms compared to the soluble form (Figure , S13).