WHAT IS IT?

Hydrogen exchange combined with mass spectrometry is a method of analyzing proteins to learn things about their structure and their dynamics.   It promises to significantly aid our understanding of proteins and protein structure in the coming years.  Traditionally, hydrogen exchange has been used in conjunction with NMR.   However, now it is also possible to use mass spectrometry to measure hydrogen exchange.

The technique began in the early 1990s with the methods capable of introducing protein molecules into a mass spectrometer for mass analysis.  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.

HOW DOES IT WORK

Some of the hydrogen atoms in proteins are capable of switching places with hydrogen atoms from the solvent molecules surrounding the protein.   If an isotope of hydrogen is used as the solvent, namely deuterium oxide, its heavier mass gets incorporated into the protein.  Because the protein now weighs more than normal, this change in mass can be monitored with high resolution mass spectrometers.

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

click for a larger image FIGURE 1

There are three kinds of hydrogens in proteins (see FIGURE 1).  Hydrogens covalently bonded to carbon (green) essentially to do not exchange.   The ones on the side chains (blue) exchange very fast and typically cannot be detected.  The ones at the backbone amide positions (red) 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.

WHAT CAN YOU LEARN?

Hydrogen exchange measurements can be used to sense changes in protein structure on a specific timescale (see FIGURE 2).  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 folding or unfolding rates Protein folding, on timescales from milliseconds to days Binding, binding constants and interacting surfaces DNA-protein interactions Several advantages of the technique include: Compared to other techniques, very little protein is required (~ 500 pmol) There is limitation on the size of the protein -- even large protein complexes can be studied Membrane proteins, nearly impossible with other techniques, can be studied

click for a larger image FIGURE 2

HOW BIG CAN YOU GO?

The size of protein or protein complex is primarily limited by how complex an experiment you want to do.  Modern mass spectrometers can measure proteins in the hundreds of thousands of daltons, even up to 1,000,000 Da.    By coupling HPLC to the mass spectrometer, complexes of many proteins can be analyzed

The largest macromolecular complexes studied so far with hydrogen exchange are viral capsids, including the capsid of P22 (19.6 MDa)! Other large things that have been studied are the capsid of the brome mosaic virus (3.6 mDa) and the GroEL/GroES complex (see reading room for references).   All of these complexes are composed of one or two subunits, repeated many times.

The most complex, large macromolecular complex studied so far is the E coli ribosome.  While smaller than some capsids, the ribosome is composed of 54 unique proteins and 3 large RNAs for a total mass of 2.5 mDa.