THE USE OF MODIFIER BUFFER IN HYDROGEN DEUTERIUM
EXCHANGE MASS SPECTROMETERY TO LIMIT THE RATE OF BACK- EXCHANGE
Muna Abdi Muse
Assessing protein structure and interactions is the foundation of structural biology.
There are several techniques to determine structure, interactions and function of proteins that are both high throughput and high resolution. Mass spectrometry (MS) is a common method used to determine the chemical properties of, Hydrogen
Deuterium-Exchange is a technique that can be combined with MS (HDX-MS).
This technique involves the exchange of labile hydrogen atoms of a polypeptide chain with deuterium atoms. HDX offers a chemical labeling strategy suitable for detecting changes in protein dynamics. Therefore, HDX is a powerful tool to monitor structure-function relationship.
In order to carry out HDX-MS the protein of interest is dissolved in a buffer containing heavy water. The amide hydrogen atoms in the protein backbone are exchanged with the deuterium atoms in the solvent and in this manner function as reporters. As a result, the site where the exchange takes place provides information regarding the structure of the protein. The precise location of the exchanged atoms can then be determined using Liquid Chromatography coupled to a Mass
Spectrometer (LC/MS).
A disadvantages of HDX is that the exchange is short lived. The incorporated deuterium atoms are lost after a short period of time; this is referred to as “Back- Exchange”. In order to minimize the rate of back-exchange and preserve the
deuterium labels and prevent their loss prior to analysis, both the temperature and pH are lowered. HDX is normally carried out at temperature near zero and under acidic condition. Despite these efforts the loss of deuterium labels is unavoidable.
Therefor, we proposed to carry out sub-zero chromatography in order to minimize the loss of deuterium labels.
To carry out sub-zero chromatography “Ethylene glycol” which is one of the main components of antifreeze is used as a buffer. By using as low as 30% of ethylene glycol as a running buffer we were able to decrease the temperature to -15°C, during analysis. For ever 5°C the temperature is decreased by, the rate of back- exchange decreases twofold.
By comparing protein labeling carried out using the standard method and this new method of sub-zero temperature, I could detect that more deuterium is incorporated during sub-zero chromatography.
