What is hydrogen deuterium exchange mass spectrometry?

What is hydrogen deuterium exchange mass spectrometry?

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful technique that can provide insights into protein behavior by serving as a link between structure, conformational dynamics and function1.

What is hydrogen deuterium exchange used for?

Only the protons present on the backbone amides are measured. The rate of hydrogen to deuterium exchange provides solvent accessibility data, which can be used to infer information on protein structure and conformation. Mass spectrometry can be used to measure the rate of deuterium uptake.

How does HDX mass spectrometry work?

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in solution. This approach relies on the fact that exposure of a protein to D(2)O induces rapid amide H → D exchange in disordered regions that lack stable hydrogen-bonding.

How do you do deuterium exchange?

Due to the acidic nature of α hydrogens they can be exchanged with deuterium by reaction with D2O (heavy water). The process is accelerated by the addition of an acid or base; an excess of D2O is required.

How much hydrogen will deuterium replace?

In the second option the alpha-carbon is the one attached to the first carbon of benzene and it contains two alpha-hydrogens hence, a total of two alpha hydrogens will be exchanged with deuterium as: Hence, maximum number of hydrogen atoms exchanged with deuterium is 5.

What is hydrogen deuterium exchange NMR?

Hydrogen–deuterium exchange (also called H–D or H/D exchange) is a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom, or vice versa. This often results in perdeuteration: hydrogen-deuterium exchange of all non-exchangeable hydrogen atoms in a molecule.

What is cross linking mass spectrometry?

Crosslinking mass spectrometry (XL-MS) analyzes protein-protein interactions that are “locked in place” to better understand how proteins affect biological processes such as signaling cascades, gene upregulation, and energy (ATP) production.

How does a mass spectrometer work?

A mass spectrometer can measure the mass of a molecule only after it converts the molecule to a gas-phase ion. To do so, it imparts an electrical charge to molecules and converts the resultant flux of electrically charged ions into a proportional electrical current that a data system then reads.

How can deuterium replace hydrogen?

Hydrogen atoms are routinely replaced with deuterium, which introduce a strong and positive scattering factor. It is often sufficient to replace only the solvent and labile hydrogen atoms in a protein crystal by vapor diffusion.

How many hydrogen will be replaced by deuterium when following compound kept in Naod d2o solution for long time?

Due to the effect of C=O. group,alpha hydrogens are highly acidic. As a result,two alpha hydrogens on left,and three alpha hydrogens on right will be replaced by deuterium.

What is the role of deuterium exchange technique in NMR spectroscopy?

Hydrogen–deuterium exchange mass spectrometry can determine the overall deuterium content of molecules which have undergone H/D exchange. Because of the sample preparation required, it is typically considered to provide an accurate measurement of non-exchangeable hydrogen atoms only.

Why is mass spectrometry used for hydrogen deuterium exchange?

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25 years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs).

How is HDX-MS mass spectrometer mass analysis performed?

Currently, the most widely used HDX-MS experimental format involves an in-solution labeling step followed by injection onto an immobilized protease column, desalting and separation of proteolytic peptides using a cooled reversed-phase LC system and finally mass analysis conducted by a mass spectrometer (Fig. 2 ).

What is the rate of hydrogen exchange dependent on?

The rate of this exchange is dependent on the folded state of the protein and its dynamics (particularly the stability of hydrogen bonding networks) and the intrinsic chemical properties of the underlying amino acid sequence 2, 3, 4.

How are proteins incubated in a deuterated buffer?

Proteins are incubated in deuterated buffer for a number of time points, allowing for the incorporation of deuterium into the protein backbone. The exchange reaction is quenched by a shift to acidic pH and a temperature drop (with the optional inclusion of denaturants and reducing agents to enhance protein unfolding).