Protease Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) is a powerful tool for studying protein structure and dynamics in structural biology and biochemistry. This technique is based on the ability of proteins to exchange hydrogen atoms in solution with deuterium atoms in the solvent. Hydrogen atoms on the surface of the protein structure or partially exposed amino acid residues can exchange with deuterium atoms in the solvent under certain conditions. HDX-MS can provide information about the three-dimensional structure, folding, interaction, dynamics, and stability of proteins.

 

Experimental Procedure

1. Hydrogen/Deuterium Exchange

The protein sample is placed in a solution containing heavy water (D₂O), allowing the exchangeable hydrogen atoms in the protein (mainly the hydrogen atoms of the amino acid side chains and the spine amino groups) to exchange with deuterium.

 

2. Termination of the Exchange Reaction

The hydrogen/deuterium exchange reaction is slowed down or stopped by rapidly lowering the pH value of the sample and lowering the temperature (usually to 0°C or lower).

 

3. Digestion

Proteins are rapidly broken down into peptides by using a protease (such as trypsin) at low temperatures and low pH. This step is performed to retain the hydrogen/deuterium exchange level as much as possible.

 

4. Mass Spectrometry Analysis

The peptides obtained by digestion are analyzed by liquid chromatography-mass spectrometry (LC-MS). In the mass spectrum, hydrogen/deuterium exchange leads to an increase in peptide mass. By measuring the change in mass, the hydrogen/deuterium exchange rate of different parts of the protein can be inferred.

 

5. Data Analysis and Interpretation

By comparing the degree of hydrogen/deuterium exchange under different experimental conditions (for example, presence or absence of ligand), insights can be gained about protein folding, dynamics, interaction interfaces, and the impact of ligand binding on protein structure.

 

Applications

1. Protein Structure and Dynamics

HDX-MS can reveal structural and dynamic information of different regions of the protein, helping to understand protein folding and movement.

 

2. Protein-Protein Interactions

By comparing changes in the pattern of hydrogen/deuterium exchange in proteins before and after interaction with other molecules, interaction areas and interfaces of the protein can be identified.

 

3. Drug Discovery

HDX-MS can be used to identify areas where small molecules interact with proteins, thereby identifying potential drug targets in drug discovery.

 

4. Stability and Conformation Changes

By analyzing the pattern of hydrogen/deuterium exchange in proteins under different conditions, their stability and conformation changes can be understood.