Structural Biochemistry/Proteins/Deuterium Exchange Mass Spectrometry (DXMS)

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Deuterium Exchange Mass Spectrometry is a powerful tool with which protein/enzyme structure and interaction can be studied. This can also determine the location and orientation of protein and enzymes associated with phospholipids.

This occurs based on the principle of hydrogen exchange with solvent. The hydrogen atoms on a protein molecule can be divided into three groups (1):

1) Hardly ever exchange (H that is attached directly to C)

2) Exchange extremely quickly (H that is attached to the side chain atom)

3) Exchange rate depends on the local environment (Amide Hydrogen N-H) (1)


The hydrogen atoms on a protein molecule that undergo exchange reaction can be followed experimentally used deuterated water.


The third process listed above, the amide hydrogen exchange is described as follows:

1) Incubated proteins in deuterated water are reacted with probes/perturbations to shows how it can influence the accessibility to water and therefore affect amid hydrogen exchange rates. It will only proceed smoothly and efficiently if the amide hydrogen is reacted in solvent water. As the name implies, hydrogens exposed to hydrophobic regions will need more time to exchange, and have an increased chance of not exchanging at all since efficiency is due to the amide hydrogen being in the solvent water.

2) The deuterium atoms can be "locked in place" to prevent further exchange (1)

3) High powered liquid chromatography-mass spectrometry analysis then uses a protease, which is a catalyst, to digest/cleave the protein into its respective peptides, which are 5-15 amino acids in length.

4) Mass spectrometry is used to fragment those digested peptides into smaller pieces, which helps in identifying the peptide.


One limitation to this approach is that collision induced dissociation causes "scrambling," where the deuterium atom changes conformation within the peptide (1). A method that reduces the chances of scrambling is electron transfer dissociation.


DXMS studies with two potent, specific, and reversible lipid inhibitors. These inhibitors provide very specific methods in docking, and when docked, the precise binding conformations of the inhibitors became defined. One of the specific and reversible inhibitors is substrate, and when this inhibitor is docked, it can provide an image of the conformation of a phospholipid molecular species bound in the active site of an enzyme (1).


DXMS could also be used in conjunction with phospholipid bilayer nanosdiscs to analyze membrane protein conformation. There have been complications in the past with studying in vitro systems by way of detergent micelles or liposomes. Detergent micelles often denature the membrane proteins due to their detergent nature. Liposomes form a mixture of small, large, and multilamellar vesicles which lead to inconsistent results (2). Nanodiscs, when used with DXMS, offers a solution to studying membrane proteins.


Nanodiscs are made with membrane scaffold protein, lipids, and the membrane protein in question mixed in a phospholipid/detergent solution (2). Detergent is removed in order for the nanodiscs to form on their own. The nanodisc allows the protein to remain in its natural conformation. The nanodiscs are then purified with size exclusion chromatogoraphy and the purified nanodiscs are subjected to the deuterated buffer multiple times(2). The DXMS needs to be carried out quickly in order to minimize deuterium loss from quenching. Three methods helped to ensure the success of the nanodiscs in conjunction with DXMS (2):

1) nanodisc disassembly by adding cholate (cholate is effective because it increases membrane scaffold protein peptides and protein digestion)

2) zirconium oxide beads separates the phospholipids from the rest of the mixture

3) optimized chromatography used to separate the membrane scaffold protein peptides from the membrane proteins peptides


DXMS in conjunction with nanodiscs allows the analysis of a protein in its natural conformation and ultimately the protein’s natural conformation leads to the understanding of its function.



Reference[edit]

1. Annu Rev Biochem. 2011 Jun 7;80:301-25. Applications of mass spectrometry to lipids and membranes. Harkewicz R, Dennis EA. Source Department of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, University of California at San Diego, La Jolla, California 92093-0601, USA. rharkewicz@ucsd.edu

2. Anal Chem. 2010 July 1;82(13):5415-5419. Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry. Hebling M, Christine.