The Principle and Application of HDX-MS Protein Structure Analysis

Protein/peptide drugs are biological macromolecules with molecular weights ranging from several thousand Da to several hundred thousand Da. Compared with traditional medicines, biopharmaceutical molecules have very complex high-level structures that are directly related to the functions of biopharmaceutical molecules.

Hvidt et al. put the protein in D2O for the first time in 1954, and used a density gradient tube to determine the hydrogen-deuterium exchange rate to study the protein structure, and thus laid the theoretical foundation for hydrogen-deuterium exchange (HDX). As a mainstream analysis method of protein structure, hydrogen-deuterium exchange mass spectrometry (HDX-MS) has been widely used.

Commonly used methods for analyzing protein structure, such as X-ray crystal diffraction, often require a lot of time to obtain protein crystals that can be used to collect data. Nuclear magnetic resonance technology is often limited by the molecular weight of the protein. Compared with the above-mentioned technology, HDX-MS has the following advantages: 1. Small sample volume requirement; 2. Fast collection and analysis; 3. Not limited by molecular weight; 4. Dynamic protein data in the natural environment.

The principle of hydrogen-deuterium exchange mass spectrometry (HDX-MS) protein structure analysis

HDX is a technology widely used to analyze the structure of protein and peptide molecules in solution. The main principle is the exchange of hydrogen atoms in the protein molecular skeleton in the solution with deuterium. The rate of exchange depends on the ionic bond of the hydrogen atom, the chemical environment, and the structure and conformation of the protein. The skeleton hydrogen atoms on the surface and the hydrogen with weak hydrogen bonding force exchange fast. In the internal structure of the skeleton hydrogen and hydrogen atoms with strong hydrogen bonding force, the exchange rate is slow. The molecular weight of the protein after hydrogen-deuterium exchange increases and the rate of the increase reflects the speed of the exchange. HDX-MS detects the quality change of protein, and then conducts the dynamic study on protein secondary, tertiary, and quaternary structures.

Application of HDX-MS technology

HDX-MS is mainly used in: 1. Research on protein folding state; 2. Protein dynamics research; 3. Analysis of protein structure changes; 4. Binding and binding site analysis of protein and ligand (small molecules, peptides, cell membranes, nucleic acids, proteins, etc.); 5. Detection of protein polymerization state.

HDX-MS analysis technology has been used in many fields of biological drug quality research. Among them, batch comparison (or primary study comparison of biosimilars) in the production of biological drugs is a more direct one. Through HDX analysis of biologic macromolecules with the same sequence from different batches or different manufacturers, software analysis of the data enables the ability to analyze at the amino acid sequence level.

The basic experimental steps of our HDX service are as follows:

The protein drug is placed in a neutral solution, under non-denaturing conditions → Add antigen and other binding molecules (optional) → Add deuterated buffer → Deuterated several time points → Terminate the reaction → Online enzymatic hydrolysis (pepsin) → LC-MS/MS analysis → Software processing data → Data mapping → Formal report.

*For Research Use Only. Not for use in the treatment or diagnosis of disease.

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