Owing to the increased spectral complexity of deuterated peptide isotopic distributions, it is typical for many peptides to be excluded during manual curation of HDX-MS data. Therefore, it is important to manually validate peptides under deuterated conditions to ensure they are suitable for subsequent HDX-MS analysis. To do this, both 1-pass and multi-pass reference peptide databases were used to search for deuterated peptide ions at a 60 second deuterium exchange timepoint acquired using a1pass (SMO, MsbA and SecYEG) or multi-pass (XylE) cIM routine (Figure ). The data was then analysed using reference peptide databases generated from undeuterated cIM 1-pass data for comparison (Table ). ) in cartoon and surface format. 1 min HDX exchange data is heat mapped onto structures as relative fractional uptake, with unsequenced regions in grey. Deuterium uptake pattern exhibits strong correlation with membrane topology.