What is it?


Hydrogen exchange (HX) combined with mass spectrometry (MS) is a method of analyzing proteins to learn things about their structure and their dynamics.  It significantly aids our understanding of proteins and protein structure. 

Some of the hydrogen atoms in proteins are capable of switching places with the hydrogen atoms from the solvent molecules surrounding the protein.  Any spectroscopic method sensitive to the differences between the isotopes of hydrogen (hydrogen, deuterium, tritium) can be used to measure exchange.  Because deuterium has a mass of 2.0141 Da and hydrogen a mass of 1.0078 Da, a mass spectrometer can be used to monitor the exchange reaction. 

In the early 1990s it became possible to introduce protein molecules into a mass spectrometer for mass analysis.  The first report of hydrogen exchange measured by mass spectrometry was:

Katta V, Chait BT (1991). Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry. Rapid Commun. Mass Spectrom. 5(4), 214.
DOI: 10.1002/rcm.1290050415.  Pubmed: 1666528.

More history can be found here. Methods development is ongoing and the technique is being applied to relevant biological problems, important proteins related to disease and to the vast number of proteins for which structural information is hard to obtain with other methods.  Our research laboratory has written extensively on the technique and some of our recent reviews and articles that describe the method and its applications are:

Wales & Engen (2006). Hydrogen exchange mass spectrometry for the analysis of protein dynamics.
Mass Spectrom. Rev. 25, 158.  DOI: 10.1002/mas.20064. Pubmed: 16208684.

Engen (2009). Analysis of protein conformation and dynamics by Hydrogen/Deuterium exchange MS.
Anal. Chem. 81, 7870.  DOI: 10.1021/ac901154s. Pubmed: 19788312.

Marcsisin & Engen (2010). Hydrogen Exchange Mass Spectrometry: What is it and what can it tell us?
Anal Bioanal Chem. 397(3), 967.  DOI: 10.1007/s00216-010-3556-4. Pubmed: 20195578. 

Engen JR, Wales TE, Shi X. (2011). Hydrogen Exchange Mass Spectrometry for Conformational Analysis of Proteins.  Encyclopedia of Analytical Chemistry, Online ISBN: 9780470027318, Wiley, Robert A. Meyers, Editor-in-Chief.  DOI: 10.1002/9780470027318.a9201.

Iacob & Engen (2012). Hydrogen exchange mass spectrometry: Are we out of the quicksand?
J. Am. Soc. Mass Spectrom. 23, 1003.  DOI: 10.1007/s13361-012-0377-z. Pubmed: 22476891.

What is it used for?

Hydrogen exchange measurements can be used to sense changes in protein structure on a specific timescale.  Some amide hydrogens, such as those at the surface of proteins, exchange very rapidly.  These hydrogens can be used to sense binding to other proteins and to analyze complexes.  Other hydrogens are buried in the hydrophobic core of the protein and may not exchange for hours, days, or even months.  Therefore the movements of proteins, and the rate of such movements can be studied.

Some of the things you can use this technique for include:

  • Protein unfolding, either natural or induced by denaturants
  • Measurement of protein motion/dynamics
  • Comparability, proper folding, complement to X-ray and NMR structures
  • Binding, binding constants and interacting surfaces
  • Epitope mapping, allosteric effects

  Several advantages of the technique include:

  • Compared to other techniques, very little protein is required (~100 pmol)
  • Size of the protein -- even large protein complexes can be studied
  • Membrane proteins, nearly impossible with other techniques, can be studied

Exchange theory

The exchange of hydrogens occurs at a specific rate, which is a function of the combination of hydrogen bonding and solvent accessibility.  By measuring hydrogen exchange rates, we can draw conclusions about the conformation and the dynamics of proteins.

There are three kinds of hydrogens in proteins.   Hydrogens covalently bonded to carbon essentially to do not exchange.  The ones on the side chains exchange very fast and typically cannot be detected.  The ones at the backbone amide positions (yellow) exchange at rates that can be measured.  Each amino acid, except proline, has one amide hydrogen.  Therefore, hydrogen exchange rates can be measured along the entire length of the protein backbone.  Additionally, the backbone amide hydrogens are involved in formation of hydrogen bonds in secondary structural elements -- both alpha helicies and beta sheets; therefore, their exchange rates are a reflection of structure and structural stability.  Watch the image above in motion with this MOVIE.  

The rate of HX depends on hydrogen bonding and solvent accessibility.  Folded proteins can have amino acids with HX rates as much as 100,000,000 times slower than the same amino acid that is not in a folded protein.  Protein folding and unfolding, whether in cells or in the test tube, represent large changes in protein structure, hydrogen bonding and solvent accessibility that can be investigated with HX MS.  Smaller structural changes critical for protein function can also be probed with HX MS. 

Even in unstructured peptides, every sequence will not exchange at the same rate. There are influences from the neighboring amino acids, which depends of course on sequence. The amino acid at the N-terminus will also exchange its backbone amide hydrogen much faster than ones further down the line. These issues have been addressed in the literature. To read more, see:

For sequence effects:
Connelly GP, Bai Y, Jeng MF, Englander SW. (1993). Isotope effects in peptide group hydrogen exchange. Proteins 17(1), 87-92.
Bai Y, Milne JS, Mayne L, Englander SW. (1993). Primary structure effects on peptide group hydrogen exchange. Proteins 17(1), 75-86.

For calculating the average deuterium loss for an average peptide:
Zhang, Z., and Smith, D.L. (1993). Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation. Protein Sci. 2, 522-531.

pH, temperature and other issues

The secret to making hydrogen exchange measurements with mass spectrometry is in controlling the variables in the experiment, especially the pH.  The rate of hydrogen exchange is very sensitive to pH -- a change in one pH unit equals a ten-fold change in the exchange rate

If the exchange rate of an average amide hydrogen in a completely unstructured peptide at pH=7 equals 10 per second, the same average amide hydrogen in the same unstructured peptide at pH=5 would have an exchange rate of 0.1 per second.  The actual time involved to make the exchange in this example is far less than 1 second.  So in the simplest case scenario, one would want to do the exchange reaction at a higher pH and then reduce the pH to stop the exchange from occurring.  This is essential to do proper HX MS, since it takes some time to carry out the mass spectral analysis.

Don't forget, proteins are not just unstructured peptides! Their exchange rates will be slowed by:
    1.  Hydrogen bonding that creates the secondary structure, primarily alpha helices and beta sheets.
    2.  Protection from the solvent, primarily due to being buried in the hydrophobic core of the protein.
    3.  Hydrogen bonding to water in the solvent, a much smaller effect than #'s 1 and 2.

The pH effect can be used in a number of ways in addition to the simplest one mentioned above.  For example, one could do protein folding at pH 7 and then pulse quickly to pH=10 to "flash-label" any amide hydrogen not involved in hydrogen bonds with other parts of the protein.

Temperature is also important. Considering the example in the Figure above, even if you do an experiment at pH 7.0 and reduce the pH to 2.5 (the minimum of the curve), there will only be about 11 minutes (half-life) for you to do the mass spectral analysis before you loose half the deuterium label. To further slow the exchange rates, the temperature must be lowered to zero degrees Celsius. At pH=2.5 and temp=0, the average half-life of exchange for the average amide hydrogen is slow enough so there is enough time to analyze the sample.  Due to the sequence effects described in the previous section above, the loss of deuterium from a labeled protein or peptide is a function of the sequence.

There are many many factors that affect the actual measurements that are eventually made with the mass spectrometer.  All of these parameters must be controlled for a successful experiment, and particularly one that is reproducible.  We have described these parameters in the following paper :

Iacob & Engen (2012). Hydrogen exchange mass spectrometry: Are we out of the quicksand?
J. Am. Soc. Mass Spectrom. 23, 1003.  DOI: 10.1007/s13361-012-0377-z. Pubmed: 22476891.


To make the measurement, mass spectrometry (MS) is used.  The following will not be an exhaustive review of 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 weights 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. General mass spectrometry geometry

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.

Mass analyzer
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.

We use mass spectrometers from the Waters Corporation.  In collaboration with Waters, we have developed an instrumental system for HX MS.  Take a tour of our lab and the instruments in the James L. Waters Mass Spectrometry Facility.