English: Illustration of an experimental workflow in hydrogen/deuterium exchange measured by mass spectrometry (HDX-MS). The black squiggly line represent a protein backbone. Blue letter "H" represent hydrogen atoms bound in the backbone amides, red letter "D" represent deuterium bound in the backbone amides. From the top, a protein solution saturated with hydrogen is diluted into concentrated deuterium oxide at which point hydrogen found in the backbone amides will exchange with deuterium from the solution. At regular timepoints (t1-t4) the exchange reaction is quenched by acidification and cooling. Then, the resulting protein sample can be subjected to digestion and desalting or sometimes gas-phase fragmentation. If the protein was digested, the resulting peptides are seperated using liquid chromatography (LC) coupled to mass spectrometry (MS). For each timepoint including a non-deuterated control (t0) the average mass of each peptide is measured. Then, while not common practice, it is possible subject each analyte to fragmentation in order to sublocalize the containing hydrogen and deuterium. To make interpretation easier, only c-type ions are illustrated in the MS/MS spectrum here. However, it is possible to observe other types of fragment ions.