It can be applied most easily to exchangeable protons and deuterons, where such a transformation occurs in the presence of a suitable deuterium source, without any catalyst.
For the backbone amide hydrogen atoms of proteins, the minimum exchange rate occurs at approximately pH 2.6, on average.
However, some methods of deuteration analysis for molecules such as proteins, are performed in aqueous solution, which means that exchange will continue at a slow rate even after the reaction is quenched.
In modern times, H–D exchange has primarily been monitored by the methods: NMR spectroscopy, mass spectrometry and neutron crystallography.
Analogous signals are not observed in 2H NMR spectra because of the low sensitivity of this technique compared to the 1H analysis.
Analysis via 13C NMR spectroscopy is also possible: the different spin values of hydrogen (1/2) and deuterium (1) gives rise to different splitting multiplicities.
Because it takes minutes to hours to record a HSQC spectrum, amides that exchange quickly must be measured using other pulse sequences.
Because of the sample preparation required, it is typically considered to provide an accurate measurement of non-exchangeable hydrogen atoms only.
[12] Pepsin, an acid protease, is commonly used for proteolysis, as the quench pH must be maintained during the proteolytic reaction.
HPLC separation of the peptic digest is often carried out at low temperature just prior to electrospray mass spectrometry to minimize back-exchange.
[13] It was proposed in 1999 that it might be possible to achieve single-residue resolution by using collision-induced dissociation (CID) fragmentation of deuterated peptides in conjunction with tandem mass spectrometry.
[17][18][20][21] More recently, ultraviolet photodissociation (UVPD) has also been investigated as a possible fragmentation technique to localize deuterium within peptides and proteins.
[22][23] The theory consolidating these apparent contradictions has to do with the dual fragmentation pathway that may arise from UV irradiation of peptides and proteins, i.e. direct and statistical dissociation.
[22] However, experimental conditions may favor statistical dissociation during UV irradiation, especially at long irradiation times and low gas pressure, leading to internal conversion of the electronic excitation energy contributed by the UV photons.
Hydrogen atoms, with between one and zero electrons in a biological setting, diffract X-rays poorly and are effectively invisible under normal experimental conditions.
Hydrogen atoms are routinely replaced with deuterium, which introduce a strong and positive scattering factor.
It is often sufficient to replace only the solvent and labile hydrogen atoms in a protein crystal by vapor diffusion.
Factors determining the time resolution of this approach are the efficiency of mixing and how quickly the quench can be performed after the labeling.
If conformation is altered as result of post-translational modification, enzyme activation, drug binding or other functional events, there will likely be a change to H/D exchange that can be detected.