Under deuterated conditions, 93% of all peptides from combined 1-pass/multi-pass peptide mapping were retained in HDX-MS readout after manual curation. Thus, this novel HDX-MS approach not only provides increased peptide identification, but also provides high quality deuterium uptake data. This is supported by the strong correlation between the observed deuterium uptake pattern and known IMP transmembrane topology (Figure and Figure ). Moreover, cIM 1-pass/multi-pass peptide mapping provided successful deuterium exchange monitoring of 220 additional peptides across all proteins relative to cIM 1-pass. For XylE and SMO, combined 1-pass/multi-pass peptide mapping produced sequence coverages at 88 and 95%, respectively, whilst this dropped to 85 and 89% when using 1-pass. XylE and SMO exhibited 30 and 31% higher redundancy, respectively. Thus, whilst the additional peptide identifications appear to favour increased redundancy, they can also help retain sequence coverage and uptake resolution under deuterated conditions. For MsbA and SecYEG, no difference in sequence coverage was observed between standalone 1-pass and combined 1pass/multi-pass. However, for MsbA, combined 1-pass/multi-pass provided a 27% increase in redundancy. For SecYEG, a more modest 5% increase in redundancy was observed. (Figure ).