On-line proteolytic digestion is then performed, followed by separation of peptides at low temperature via reverse phase liquid chromatography (LC) and detection via mass spectrometry (MS). This "bottomup" LC-MS approach allows changes in HDX-MS readout to be localised to specific structural regions of the protein at peptide-level resolution . Therefore, the quality of bottom-up HDX-MS data is largely determined by the number of peptides that are identified and monitored throughout an LC-MS experiment.