Hydrogen–deuterium exchange, also called H–D or H/D exchange, is a chemical reaction, which can be used most easily to exchangeable protons and deuterons without any catalyst but in the presence of a suitable deuterium source. This often causes perdeuteration: hydrogen-deuterium exchange of all non-exchangeable hydrogen atoms in a molecule. It has built itself as a strong method for amide hydrogen/deuterium (H/D) exchanging of proteins monitored by mass spectrometry, which is for probing protein conformational dynamics and protein interactions. In the past 10 years, H/D exchange mass spectrometry has developed to an adaptable technique for detecting conformational dynamics and interactions in proteins, protein-ligand and protein-protein complexes. HX MS detects solution conformation so crystallization is not required, it requires very little sample, for example, 500–1,000 picomoles for an entire experiment, it is subject to studying proteins that are difficult to purify or that can only be handled at low concentrations and it can show the changes to conformation and dynamics on a wide timescale.
Figure 1: Overview of amide hydrogen exchange in proteins
The improvement of HX MS experiment also rely on the advances in liquid chromatography and mass spectrometry. However, there exists a major limitation in terms of chromatography during HX MS experiments is that separation must be done at 0 °C while the chromatographic efficiency in traditional HPLC is relatively poor. New separation media and the use of UPLC are solving this issue and allowing for analysis of a growing number of proteins and protein systems. Historically, the large amount of data generated during hydrogen exchange experiments have made data processing time-consuming. There is one key parameter of an HX MS experiment to obtain the protein to be studied. If the proteins are scarce, difficult to overexpress or obtain in the concentrations needed for some biophysical methods, there may be major problems obtaining suitable material for analysis. Luckily, HX MS has some of the lowest requirements of any of the biophysical techniques that can provide conformational data, so it can access some of these difficult-to-handle proteins, especially an estimated 30% of eukaryotic proteins that contain unstructured regions.
Creative Proteomics has a strict workflow to analyze Hydrogen Deuterium Exchange Mass Spectrometry to meet your requirements.
