Hydrogen exchange mass spectrometry: what is it and what can it tell us?
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Proteins are undoubtedly some of the most essential molecules of life. While much is known about many proteins, some aspects still remain mysterious. One particularly important aspect of understanding proteins is determining how structure helps dictate function. Continued development and implementation of biophysical techniques that provide information about protein conformation and dynamics is essential. In this review, we discuss hydrogen exchange mass spectrometry and how this method can be used to learn about protein conformation and dynamics. The basic concepts of the method are described, the workflow illustrated, and a few examples of its application are provided.
KeywordsDeuterium Protein mass spectrometry Protein dynamics Protein conformation
Proteins play a pivotal role in most biological processes, including DNA replication, cell division, cell death, immune response, and cellular signal transduction. Many of these processes are thought to be carried out by protein machines containing at least 10 or more proteins . To fully understand how proteins drive and contribute to basic biological and biochemical events, techniques that probe the fundamental properties of proteins are necessary. Not only must these methods provide information about protein function, they must also help reveal how function is tied to protein conformation and dynamics. In the past 60 years or so, there have been profound advances in the techniques for protein analysis, including such Nobel Prize-winning methods as nuclear magnetic resonance (NMR), X-ray crystallography, small angle X-ray scattering, and cryo-electron microscopy. As with everything, each of these techniques has advantages and disadvantages for studying protein conformation and dynamics, and often a combination of methods is required for full protein understanding. An orthogonal biophysical technique that has also seen profound advances in the last 20 or so years is Hydrogen Exchange monitored by Mass Spectrometry (HX MS). HX MS probes solution conformation so crystallization is not required, it requires very little sample (500–1,000 picomoles for an entire experiment), it is amenable to studying proteins that are hard to purify or that can only be handled at low concentrations (as low as 0.1 µM), and it can reveal changes to conformation and dynamics on a wide timescale (for a recent review and perspective, see Ref. ).
Overview of hydrogen exchange: what is it?
There are various factors that affect hydrogen exchange in proteins . The four primary factors are pH, temperature, solvent accessibility, and hydrogen bonding. Temperature and pH can be controlled experimentally meaning that the rate and location of exchange then becomes a function of hydrogen bonding and solvent accessibility. While amide hydrogens of fully solvent-exposed peptides at pH 7 exchange very rapidly with rates of 10–1,000 s−1 (depending on solvent conditions, etc.) , in folded proteins amide hydrogens display a variety of exchange rates depending on their position within the protein and whether they are involved in intramolecular hydrogen bonding. Figure 1b illustrates how solvent accessibility and hydrogen bonding relate to exchange in proteins. Regions that are highly dynamic and solvent-exposed (like the loops connecting the alpha helices) will exchange rapidly while regions that are less dynamic (i.e., “rigid”) and/or involved in hydrogen bonding networks or buried within the interior of the protein (such as β-sheets or α-helices) will exchange slower. In folded proteins some amide hydrogens exchange quickly while others exchange much slower on timescales from minutes to months [4, 6]. Note also that the backbone amide hydrogens are the ones participating in the hydrogen bonds that hold α-helices and β-sheets together. If there is a change to the solvent accessibility or the hydrogen bonding network of a protein, the rate and location of deuterium incorporation can be altered.
Monitoring hydrogen exchange
The different chemical properties of hydrogen and deuterium allow several biophysical techniques to distinguish between the two isotopes. If deuterium incorporation into proteins is monitored with NMR, for example, the amide proton peaks (from hydrogen) disappear as the protein becomes deuterated because deuterium is NMR silent. As the mass of hydrogen is 1.0078 Da and the mass of deuterium is 2.0141 Da, deuterated proteins will have a larger mass than non-deuterated proteins . All that remains to convert deuteration information into conformational information is to determine how fast a protein is deuterated and where the deuterium goes.
What can hydrogen exchange tell us?
HX MS can provide very useful information, including: how conformation changes upon binding, protein folding and unfolding pathways, glimpses into the structure of proteins that will not crystallize or are not amenable to NMR, which regions of a protein are solvent-exposed and dynamic, location and properties of binding sites and surfaces, dissociation constants and measures of protein stability under various conditions. The raw mass spectra of the mass of the protein, or each peptide from the digestion, are converted into a deuterium uptake plot where the level of deuterium is plotted versus the labeling time. Deuterium uptake plots for both intact analysis and peptide analysis contain information about how solvent-exposed and dynamic a protein is in solution. For a detailed explanation of all the information that can be derived from deuterium uptake plots see Ref. .
In addition to probing structural changes in proteins as a result of ligand/inhibitor binding, HX MS can be used to study many other types of protein interactions, including protein–protein interactions. Since the majority of cellular processes are orchestrated by multiple proteins and large macromolecular protein complexes, being able to study the structural consequences of these interactions is extremely important. Figure 3b illustrates an example of how HX MS could be used to study protein–protein interactions. In this hexameric protein, exchange can be compared in the isolated monomer versus the monomer in the assembled hexamer (Fig. 3b(i)). Then, with the use of pepsin digestion, the specific regions affected by complex formation can be determined (residues 10–28 are involved in hexamer formation, residues 50–68 are not; Fig. 3b(ii and iii)).
One could imagine many protein–protein interactions in which determining the effects and location(s) of changes in HX upon binding would be useful. For example, HX MS has been used to study the structural changes induced by pH changes in the capsid protein of the brome mosaic virus  and it has been used to investigate conformational changes in the HIV-1 capsid protein as a result of HIV assembly and maturation . The large size and complexity of viral capsids make them challenging to study by many structural means, particularly when detailed information about conformational changes is desired. HX MS, however, can allow access to such information and has therefore become important as a tool for probing large proteins and protein complexes.
Because the function of proteins is dictated by their structure and movements in solution, any biophysical technique which allows for the molecular investigation of proteins will be invaluable to aiding in our understanding of these molecular machines. Amide hydrogen exchange monitored by mass spectrometry is just one such technique for studying proteins. To become robust and high throughput, HX MS faces some challenges. Development of robotics and total automation of sample handling and data processing will make the method more amenable to high-throughput types of studies. Advances in liquid chromatography and mass spectrometry will also continue to improve the HX MS experiment. A major limitation in terms of chromatography during HX MS experiments is that separation must be done at 0 °C where chromatographic efficiency in traditional HPLC is relatively poor. New separation media and the use of UPLC  are addressing this issue and also allowing for analysis of bigger and bigger proteins and protein systems . The large amount of data produced during hydrogen exchange experiments have historically made data processing time-consuming. Reducing the data to a form that is easily understood in terms of conformation for a given protein can be arduous. Recent software developments (e.g., HYDRA , HD Desktop , and TOF2H ) significantly reduce the burden of HX MS data analysis although much more work needs to be done in this area. More sensitive mass spectrometers will also improve HX MS. One important parameter of an HX MS experiment is obtaining the protein to be studied. If proteins are rare, difficult to overexpress or obtain in the concentrations needed for some biophysical methods, or just otherwise uncooperative, there may be significant problems obtaining suitable material for analysis. Luckily, HX MS has some of the lowest requirements of any of the biophysical techniques that can provide conformational data, thereby providing access to some of these proteins that are difficult to deal with, particularly the estimated 30% of eukaryotic proteins that contain unstructured regions . We envision that conformational data on many proteins that were considered impossible to analyze will be provided by HX MS in the future.
We would like to thank Dr. Thomas Wales and Dr. Thomas Smithgall for the Lyn SH3:Nef binding data. This work was supported in part by the National Institutes of Health (R01-GM070590 and R01-GM086507). This is contribution 959 from the Barnett Institute.
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