An Overview of H/D-Exchange
Mastering the art and science of H/D-Exchange Mass Spectrometry to gather precise feedback on lead candidates
ExSAR utilizes proprietary platform amide hydrogen/deuterium exchange mass spectrometry technology (also referred to as H/D-Exchange) to facilitate drug development and target validation. ExSAR’s H/D-Exchange platform consists of automated sample handling and proprietary software to generate rapid and reproducible measures of amide proton exchange rates. Typical applications involve drug binding interface determination and the detection of alterations in protein structure following protein/protein or protein/ligand complex formation. Other H/D-Exchange applications focus on the detection of structure alterations in response to environmental modifications (e.g., new formulation, new buffer, etc.) or following genetic mutation (e.g., wild-type vs. inherited mutation).
The science of H/D-Exchange begins with a couple of chemistry basics. The first is a simple molecule of water (H2O), which of course, consists of two hydrogen (H) atoms and one atom of oxygen (O). The second is the fact that hydrogen atoms are more or less equally distributed throughout proteins. When we take a closer look at some of these hydrogens, such as backbone amide hydrogens, we observe that they engage in exchange with solvent hydrogens. Further, the exchange takes place in a timeframe amenable to analysis.
On transfer from an H2O-based solvent system to one primarily consisting of D2O, a form of water containing the heavier isotope of hydrogen (deuterium or D), exchange of H for D atoms over time result in an increase in protein mass. The likelihood that a particular amide will undergo exchange is largely a function of protein structure and solvent accessibility. Molecular motions present in all proteins, collectively referred to as protein dynamics, facilitate solvent accessibility by momentarily changing or disrupting protein structure. The rates of amide H/D-Ex reflect the conformation of the protein. Within folded domains, amide hydrogen exchange rates often vary by eight orders of magnitude. Folded regions are readily distinguished from unfolded regions. The intrinsic sensitivity of H/D-Ex to the amide environment in the protein allows an accurate assessment of protein conformation, and stability.
ExSAR follows hydrogen/deuterium exchange by mass spectrometry (HXMS). Using the shift in protein mass as a marker, we can analyze how protein targets respond to the drugs that bind them.
The exchange rate of each backbone amide hydrogen is unique to its environment; disordered regions and/or surface exposed regions exchange fast, ordered and/or buried regions exchange slow. Following an incubation period, the exchange reaction is essentially quenched by shifting the pH to around 2 while lowering the temperature to near 0°C. The exchanged protein is then proteolyzed with acid stable proteases, such as pepsin. The peptic fragments are then chromatographically separated and their masses determined by mass spectrometry. The experiment is repeated in the absence of deuterium and the weight gain of each fragment attributed to deuteration.