Hydrogen Deuterium Exchange (HDX)
Mass Spectrometry (MS)
The following is not an exhaustive review of mass spectrometry (MS) – please seek other sources for more MS theory and background. Mass spectrometry is invaluable for measuring the molecular weight of proteins. Typical analysis can identify the molecular weight of a 20,000 kD protein to within +/- 2.0 daltons while analysis of peptides can be even more accurate. It is for these reasons that mass spectrometry is used in the Engen laboratory to investigate the incorporation of hydrogen into proteins during hydrogen exchange. Because deuterium weighs 2.0141 Da and hydrogen weighs 1.0078 Da, deuterated and non-deuterated proteins and/or peptides can easily be distinguished.
This a simple diagram of a mass spectrometer. It can be divided into three parts: ionization, mass analyzer, and detector.
Proteins and peptides must first be converted to the gas phase via ionization. Ionization of proteins/peptides can be done in several ways. MALDI (matrix assisted laser desorption mass spectrometry) uses a laser beam to zap crystallized protein/matrix mixtures into the gas phase. ESI (electrospray ionization) converts a liquid-protein solution into fine droplets. The solvent is evaporated from the fine droplets and charge is deposited on the protein molecules. ESI can be coupled to chromatography, such as HPLC and UPLC. As peptides/proteins elute from a chromatography column they are sent directly into the mass spectrometer where they are ionized.
Once proteins/peptides are ionized, they must be separated according to their molecular weight. There are several types of mass analyzers: magnetic sector, quadrupole, ion trap, time-of flight.
- Magnetic sector: uses a magnetic field to separate
- Quadrupole: uses a combination of RF fields and voltage to separate
- Ion trap: a 3D quadrupole, uses RF and electric fields to separate
- Time-of-flight: separates with time. Heavier molecules take longer to fly down a tube than lighter molecules
All mass analysis is done in a vacuum so that molecules do not collide with each other. Sometimes, molecular collisions are desirable and can be used to fragment larger molecules into smaller ones. This procedure is called MS/MS.
After a collection of proteins/peptides have been separated according to mass, they must be detected. Ions can be detected with electron multipliers or with diode array detectors.