Hydrogen–deuterium exchange

Hydrogen–deuterium exchange (also called H–D or H/D exchange) is a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom, or vice versa. 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. The use of acid, base or metal catalysts, coupled with conditions of increased temperature and pressure, can facilitate the exchange of non exchangeable hydrogen atoms, so long as the substrate is robust to the conditions and reagents employed. This often results in perdeuteration: hydrogen deuterium exchange of all non exchangeable hydrogen atoms in a molecule. An example of exchangeable protons which are commonly examined in this way are the protons of the amides in the backbone of a protein.

In protic solution exchangeable protons such as those in hydroxyl or amine group exchange protons with the solvent. If D2O is solvent, deuterons will be incorporated at these positions. The exchange reaction can be followed using a variety of methods (see Detection). Since this exchange is an equilibrium reaction, the molar amount of deuterium should be high compared to the exchangeable protons of the substrate. For instance, deuterium is added to a protein in H2O by diluting the H2O solution with D2O (e. g. tenfold). Usually exchange is performed at physiological pH (7.0–8.0) where proteins are in their most native ensemble of conformational states. The major drawback is that it requires a prior assignment of the spectrum for the protein in question. This can be very labor intensive, and usually limits the method to proteins smaller than 25 kDa. Because it takes minutes to hours to record a HSQC spectrum, amides that exchange quickly must be measured using other pulse sequences.

Hydrogen–deuterium exchange mass spectrometry (HX MS or HDX MS) can determine the overall deuterium content of molecules which have undergone H/D exchange. Because of the sample preparation required, it is typically considered to provide an accurate measurement of non exchangeable hydrogen atoms only. It can also involve H/D exchange in the gas phase or solution phase exchange prior to ionization. It has several advantages in NMR spectroscopy with respect to analysis of H–D exchange reactions: much less material is needed, the concentration of sample can be very low (as low as 0.1 uM), the size limit is much greater, and data can usually be collected and interpreted much more quickly. However, by careful optimization of instrument parameters which cause ion heating, hydrogen scrambling can be minimized to a degree which preserves the solution phase isotopic labeling until fragmentation can be performed using a technique where scrambling does not occur. More recently, ultraviolet photodissociation (UVPD) has also been investigated as a possible fragmentation technique to localize deuterium within peptides and proteins. In this regard, the conclusions have been mixed, while it is possible to obtain UVPD fragments which has not undergone scrambling under certain conditions, others have shown that scrambling can occur for both peptides and proteins during the UVPD fragmentation step itself. That is, if experimental conditions favor direct dissociation and the precursor ion is kept at low internal energies before and during fragmentation the deuterium level of the resulting fragments will correspond to the non scrambled precursor. and protein–protein interactions.

HDXsite is an online websever which includes some applications such as HDX modeller increasing the resolution of experimental HDX data and modeling protection factors for individual residues.