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    Hydrogen-Deuterium Exchange Mass Spectrometry Experiment

      With the advancement of biopharmaceutical technology, proteins, as the main targets or therapeutic agents in drug research, are gradually being applied to various clinical indications. The structure of a protein is dynamic and changes with post-translational modifications, ligand binding, or interactions between proteins. Therefore, studying the changes in the high-level structure of proteins in the drug design process is crucial for understanding the mode and function of potential drugs.


      Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a mass spectrometry technique for studying the spatial conformation of proteins. The principle is that when proteins are diluted with heavy water, the unstable hydrogen atoms in protein molecules exchange with the deuterium atoms in heavy water, and the mass shift of peptides is monitored by mass spectrometry. There are three types of hydrogen atoms in protein structures.The first is hydrogen that is covalently bonded to the protein's carbon skeleton, which cannot undergo hydrogen-deuterium exchange reactions.The second is the side chain hydrogen of the protein, which has an extremely fast exchange rate and is difficult to monitor under solution conditions.The third is the protein backbone amide hydrogen (marked in yellow in the figure, except for proline), which has a moderate exchange rate and is closely related to pH, temperature, protein's three-dimensional structure, and conformational dynamics. Therefore, HDX-MS experiments usually monitor the exchange behavior of this part of hydrogen.


      Five Basic Steps of HDX-MS Experiment

      1. Sample Hydrogen-Deuterium Exchange Reaction

      the prepared protein aqueous solution is diluted 15 to 20 times with a heavy water solution for the hydrogen-deuterium exchange reaction. A high concentration of heavy water (over 90%) ensures that the reaction mainly exchanges from hydrogen to deuterium.


      2. Termination Reaction

      The reaction is terminated by lowering the pH value of the sample solution to 2.5 and the temperature to 0°C.


      3. Sample Digestion

      Under termination conditions, enzymatic digestion is carried out with immobilized protease (5 to 10 min) to obtain a large number of deuterated peptides.


      4. Mass Spectrometry Detection

      Deuterated peptides are separated by low-temperature HPLC/UPLC, and then entered into MS analysis. Based on the mass difference between hydrogen and deuterium, MS can monitor HDX changes by measuring the mass center displacement of peptides.


      5. Data Analysis

      By processing and analyzing mass spectrometry data, information such as the hydrogen-deuterium exchange rate and protection level of proteins can be obtained. This information can be used to infer the structure and conformational changes of proteins, as well as the interactions between proteins and other molecules.


      Compared to classical protein structure research methods (X-ray crystal diffraction and nuclear magnetic resonance, etc.), HDX-MS cannot provide an accurate protein spatial structure. The main information it directly provides includes which amino acid sequences are located on the surface of the protein's spatial structure (including those in dynamic changes), potential active sites, and protein-protein interaction sites, etc. However, HDX-MS has advantages that other classical methods do not have: (1) It can conduct research on the dynamic changes of protein structures, including active sites and epitopes in changes; (2) It has unique advantages in the study of protein complex conformations; (3) It has small sample demand, relatively low purity requirements, and the research object is the natural conformation of the protein under a solution environment rather than the conformation in the crystal.


      As an important biochemical analysis technique, the hydrogen-deuterium exchange mass spectrometer experiment has been widely used in the research of protein structure, structural dynamic changes, and protein interaction, forming a complement to the classical protein structure research methods. MtoZ Biolabs uses waters' advanced nanoACQUITY UPLC HD-Exchange System analysis system, combined with sub-two nanometer chromatographic particle fillers, can drastically shorten the liquid analysis time requirement without compromising the chromatographic separation effect, and has an ultra-high degree of separation. In addition, MtoZ Biolabs uses an online automatic preprocessing and high-resolution mass spectrometer in combination, which not only ensures the reliability of experimental data but also improves experimental efficiency, fully guarantees preprocessing results and identification result reliability. You only need to tell us your experimental purpose and send us your samples, and we will be responsible for all subsequent project matters, including sample processing, enzyme cutting, mass spectrometry detection, original data analysis, etc.

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