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Review
. 2010 Jun;397(3):967-72.
doi: 10.1007/s00216-010-3556-4. Epub 2010 Mar 1.

Hydrogen exchange mass spectrometry: what is it and what can it tell us?

Sean R Marcsisin  1 , John R Engen
Affiliations
Review

Hydrogen exchange mass spectrometry: what is it and what can it tell us?

Sean R Marcsisin et al. Anal Bioanal Chem. 2010 Jun.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Overview of amide hydrogen exchange in proteins. a Part of an amino acid sequence is shown. All backbone nitrogens have an amide hydrogen (blue), except the nitrogen in proline (brown), thereby providing a sensor at each amino acid along the length of the protein chain. The backbone amide hydrogens (blue) are in continuous exchange with hydrogens in the solvent. Some hydrogen atoms located within the amino acid side chains (R1, R2, etc.) also become deuterated; however, these positions revert back to hydrogen during the liquid chromatography (LC) step of the experiment. Hydrogen bonded to carbon does not exchange. b Once placed in a solution of D2O, backbone amide hydrogens will become deuterated. The exposed and “dynamic” regions of proteins will exchange rapidly while protected and “rigid” regions of proteins will exchange slower. The rate of exchange for any given amide hydrogen is dictated by pH, temperature, solvent accessibility, and hydrogen bonding. The temperature and pH are controlled during an HX MS experiment leaving the structurally related parameters of solvent accessibility and hydrogen bonding as the governing factors for deuteration
Fig. 2
Fig. 2
Workflow of a typical HX MS experiment. Protein samples are equilibrated at the desired temperature and pH, in a buffer compatible with the protein. Protein solutions are then diluted (typically 10-fold or more) with the identical buffer containing 99.9% D2O instead of H2O. The exchange reaction proceeds for various amounts of time and is quenched by lowering the pH to 2.5 and the temperature to 0 °C. The pH and temperature adjustment reduces the amide exchange rate to its minimum. Deuterated, quenched protein can then be either directly injected into a mass spectrometer for mass analysis or digested with an acid protease prior to liquid chromatography and mass analysis. The mass spectra are analyzed and the uptake of deuterium over time determined and plotted, either for the intact protein, or for each of the peptic peptides
Fig. 3
Fig. 3
HX MS reports on the effects of protein binding: a protein plus small molecule inhibitor or b protein–protein interactions as a result of quaternary structure formation. In a, analysis of the intact, undigested protein (i) can reveal if there are any global conformation and/or dynamic changes as a result of inhibitor binding. In this example, the inhibitor causes a reduction in deuterium incorporation (red curve). By digesting the deuterated protein with a protease, one can observe the location of the differences (ii and iii). Residues 20–35 of this protein (ii) undergo a reduction in deuterium incorporation upon inhibitor binding while residues 70–89 (iii) do not. The location of each peptide is known (structural insets). In b, deuteration of the monomeric subunit of a hexameric protein was compared with deuteration of the same monomer when part of the hexameric assembly found in vivo. Intact protein analysis (i) indicates protection from exchange in the hexamer and the location was determined to be primarily residues 10–28 (ii) and not residues 50–68 (iii)
Fig. 4
Fig. 4
Using hydrogen exchange to monitor the HIV-1 Nef:Lyn SH3 complex. a An advantage of HX MS over some other biophysical techniques is the ability to detect different protein populations in solution. In this example, the two populations appear in the mass spectra: one representing the folded state (blue distribution) and the other representing the unfolded state (red distribution). The appearance of two distributions occurs when the rate of interconversion of the two populations (i.e., folded and unfolded) is slower than the amide exchange rate (recently discussed in Ref. [18]). If a molecule unfolds, it will become totally deuterated, hence the higher mass. b,c The Src Homology 3 (SH3) domain of the Lyn kinase shows a bimodal pattern in intact protein HX MS (+6 charge state shown). This pattern indicates partial, cooperative unfolding as described in a. One of the labeling timepoints captures the populations at an approximate 60:40 ratio (folded to unfolded), indicated by the asterisk. The actual 50:50 population point (or t1/2 for the unfolding reaction) is observed 12 min after deuterium labeling begins. In c, the unfolding is shown for the +6 charge state of intact Lyn SH3 bound to the HIV-1 accessory protein Nef [22]. Notice that while the pattern of the bimodal distribution of folded and unfolded species appears similar to that shown for Lyn SH3 (b), the time at which it appears during the deuterium labeling time course is much longer in the bound form (c). The 60:40 ratio is again marked with an asterisk and occurs at a much later time in the labeling timecourse (30 mins). Data such as these give insight into the molecular mechanisms which govern enhancement of HIV replication through the Nef:Lyn complex
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