Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Abstract

Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

Figure 1

(A) Overview of the basic principles of amide exchange in proteins. The protein samples a closed (cl) and open (op) forms, of which only the open form is accessible for deuterium exchange. The relative rates of opening (k_op), closing (k_cl), and the chemical exchange rate of the accessible amide (k_ch) govern whether the experimental observed rates will fall in the realm of (B) EX1 kinetics or (C) EX2 kinetics. (D) For EX2 kinetics, the free energy of the local stability can be calculated using the ratio of the k_ch and the observed rate k_obs, a ratio referred to as the protection factor (PF) associated with the local structure.

Figure 2

(A) Deuterium uptake at pulse times in D₂O is plotted across a range of pH. (B) Exchange rates as a function of pH show the transition point from the EX2 to and EX1 exchange regime. Above pH 11, the exchange reaches the EX1 limit where k_ch ≫ k_op and the rate is entirely governed by the opening rate of the amide. Reproduced with permission from ref (4). Copyright 2006 American Chemical Society.

Figure 3

Examples of mass spectra at various time points showing the isotopic distributions resulting from pure EX2 (A), pure EX1 (B), a noninterconverting mixture of conformers (C), mixed EX1/EX2 (D), and artifactual bimodal spectra attributed to sample carryover (E). The minor subpopulation in (C) is denoted with a *. The arrow in (E) reflects the contribution to the signal from a previous injection. Reproduced with permission from ref (665). Copyright 2016 Elsevier.

Scheme 1

Scheme 2

Scheme 3

Figure 4

Rate of exchange of an unstructured amide is shown as a function of pH. The gray line is the summed rate from the acid and base catalyzed exchange contributions, which are individually depicted with the red and blue dashed lines, respectively. The black line above the hatch marks is the net rate accounting for the contribution of water catalysis. Reproduced with permission from ref (71). Copyright 2012 American Chemical Society.

Figure 5

k_ch of polyalanine is plotted as a function of temperature based on calculations from Bai et al.

Figure 6

Relationship between solution pressure and pH for phosphate (white) and MOPS (black) buffer are shown. Circles, squares, and diamonds reflect a starting solution pH of 7.0, 7.5, and 8.0, respectively. Reproduced with permission from ref (92). Copyright 2005 Elsevier.

Figure 7

Relationship between the salt concentration and pH for triethanolamine (TEA) buffer and citrate buffer (CA) are shown. The salts used in the study were tetramethylammonium chloride (▷), choline chloride (◇), cesium chloride (○), potassium chloride (▽) sodium chloride (□), and lithium chloride (△). Solid lines are predictions based upon extended Debye–Huckel equation using ionic size parameter 4 × 10^–10 m. Reproduced with permission from ref (85). Copyright 2006 American Chemical Society.

Figure 8

Predicted relationship between k_ch and pH at different levels of organic solvent. The general shift in the position of the “V” shaped curves results from offsets to solution conditions and the lower concentration of water available for catalyzing amide exchange. Reproduced with permission from ref (8). Copyright 1985 Elsevier.

Figure 9

Amide exchange kinetics in proteins vary over 8 orders of magnitude. Comparative studies that sample only a limited temporal range can lead to missed information. Plots a–d show various kinetics for two states of a protein (red and green). Only in plot a is the comparison truly the same for all exchange times. Plots b–d have actual difference in exchange rates, but they are invisible due to the limited temporal sampling (highlighted region of the plot). Reproduced with permission from ref (24). Copyright 2017 American Chemical Society.

Figure 10

Example peptide spectra as undeuterated, 0% (in-exchange control), after 10 s labeling in D₂O, after 10 h of labeling in D₂O, and 100% exchanged control (top to bottom). The quality control in the middle is used to verify that the protein has not been perturbed during the 10 h incubation by verifying that the spectra looks identical to the first 10 s time point.

Figure 11

Deuteration levels are shown throughout a 5 min digestion step and a 10 min LC step for peptides starting with 0% (tan), 50% (blue), and 100% (red) deuterium labeling. Final values recorded by the experiments are shown to the far right with the same coloring. Rates were estimated from Bai et al.

Figure 12

Comparison of peptides for aprataxin and PNKP-like factor (APLF). Each bar under the primary sequence shows a peptide from either pepsin (red) or NepII (blue) digest. Sites indicated with triangles show sites that are uniquely cleaved by pepsin (red) or NepII (blue). Reproduced with permission from ref (137). Copyright 2015 American Chemical Society.

Figure 13

Comparison of peptides obtained from pepsin digest of HIV-1 capsid mutant protein at either ambient pressure (black) or >9000 psi (red). Lines represent start and end positions for observable peptides. Reproduced with permission from ref (138). Copyright 2010 American Chemical Society.

Figure 14

Chemistry or disulfide bond reduction using TCEP.

Figure 15

Peptic coverage maps are shown for nerve grown factor β using either (a) TCEP for reduction or (b) electrochemical reduction prior to pepsin digestion. Bars under the primary sequence show each unique peptide observed. Cysteine residues involved in disulfide bonds are shown in red. Figure adapted from Trabjerg et al.

Figure 16

Schematics of the plumbing system for analyzing HDX samples based on Wang, Pan, and Smith. Samples are injected onto a loading loop through the injection valve (left). The loading pump flows digestion buffer to push the sample over the pepsin column onto a peptide trap column. The analytical valve (right) is then toggled so the gradient elutes peptides from the trapping column through the analytical column and out.

Figure 17

(A) Comparison of UPLC separations at either 0 °C, −10 °C, or −20 °C showing the total ion chromatogram. (B) Deuterium levels across different peptides are shown for each temperature (top) and the difference between predicted levels of deuterium vs measured (below). Reproduced with permission from ref (190). Copyright 2017 Elsevier.

Figure 18

CE-MS (top) and LC-MS (bottom) separation of a bovine hemoglobin pepsin digest. Reproduced with permission from (197). Copyright 2015 American Chemical Society.

Figure 19

Example illustrating the benefits of high mass resolution. The overlapped spectra contains two deuterated peptides (blue, top; red, bottom) and some of the isotopic peaks closely overlap in m/z (inset). Without the high mass resolution, these isotopic peaks would confound accurate measure of deuterium uptake for both peptides. Reproduced with permission from ref (216). Copyright 2010 American Chemical Society.

Figure 20

Detector saturation can lead to distorted isotopic envelopes that offset deuterium measurements. An undeuterated peptide was analyzed by TOF (cyan, bottom), showing the expected isotopic distribution. The same peptide analyzed using an additional ion mobilty separation leads to detector saturation distorting the isotopic profile (gray, middle). The dashed lines indicate the expected isotopic profile. Using a dynamic range extension feature to account for the large ion flux largely mitigates the distortions attributed to detector saturation (magenta, top). Reproduced with permission from ref (215). Copyright 2017 American Chemical Society.

Figure 21

Comparison of the sequence coverage for V2R either without (a) or with (b) parallel proteomic analysis to map out PTMs. The dashed segment contains phosphorylation sites, which were identified from trypsin digested samples and subsequently used to identify two pepsin peptides from this region (c). Reproduced with permission from ref (225). Copyright 2019 American Chemical Society.

Figure 22

Depiction of deuterium scrambling in a partially deuterated peptide. The deuterium is localized to only the c-terminal half, but upon collision activation, the protons and deuterium migrate along the exchangeable sites on the peptide, leading to loss of the deuterium localization (“scrambling”). With electron capture dissociation, there is very little ion excitation, which leaves the deuterium in place to provide informative fragment ions. Reproduced with permission from ref (241). Copyright 2008 American Chemical Society.

Figure 23

Site-specific determination of amide exchange kinetics using ETD. Amide exchange within peptide 67–82 of cytochrome c were analyzed from the available c (left) and z (right) ions, indicated by ×s. As a reference, the predicted deuterium levels in the absence of scrambling (blue) and complete scrambling (red) are shown. Reproduced with permission from ref (254). Copyright 2017 American Chemical Society.

Figure 24

UVPD of deuterated peptides can occur without scrambling. Deuterium content of various a ions are shown at conditions that prevent (top) or induce scrambling (bottom). The uptake measured is plotted in blue. Theoretical positions of the deuterium levels assuming no (green) or full (red) scrambling are indicated in dashed lines. Reproduced with permission from ref (258). Copyright 2018 American Chemical Society.

Figure 25

Example of top-down HDX-MS to study the dynamics of histone tails. The deuterium uptake for the c15 ion (left) and z74 ion (right) generated by ETD are shown for each deuterium exchange time point and the fully deuterated (FD) control. Reproduced with permission from ref (275). Copyright 2018 Elsevier.

Figure 26

Top-down analysis of the various phosphorylated states of calmodulin. The top spectrum shows the unphosphorylated calmodulin (CaMp0) and four different phosphorylated proteoforms. The bottom spectrum is after 20 s of deuterium exchange. Each proteoform could be mass-isolated and the amide exchange characterized with high spatial resolution using ETD. Reproduced with permission from ref (271). Copyright 2016 from Elsevier.

Figure 27

Middle-down HDX-MS was used to study herceptin. Limited pepsin digestion yielded three large fragments (1–3) that were resolved and independently analyzed by ETD to increase the sequence coverage that was obtained from direct top-down ETD analysis. Figure adapted from ref (277).

Figure 28

Comparison of ETD (top) and UVPD (bottom) fragmentation used for high resolution top-down HDX-MS. Each notch in the sequence represents a fragment ion that was observable for HDX-MS. Reproduced with permission from ref (257). Copyright 2018 American Chemical Society.

Figure 29

Epitope mapping of three monoclonal antibodies to Staphylococcus aureus manganese transporter protein (MntC). Regions in yellow exhibited slowed exchange when bound to the antibody. Figure adapted from ref (284).

Figure 30

Allosteric changes observed in the Nipah virus G ectodomain upon binding ephrinB2. Regions of the G ectodomain that do not change (white) become more protected (blue) or less protected (red) are indicated on the structure. The N-terminal stalk helices that also become less protected are shown in the bottom with their predicted positions. The position of EprhinB2 is shown in yellow. Figure adapted from ref (300).

Figure 31

Mechanism of action of Mab SRK-015 on the inhibition of myostatin. Antibody binding to the distal arm of myostatin elicits allosteric effects throughout the protein including protection around the furin cleavage site to limit its accessibility for proteolytic cleavage. Figure adapted from ref (301).

Figure 32

Pulse labeling HDX-MS was used to study the folding kinetics of cytochrome c. Fully deuterated unfolded protein was mixed with D₂O from 5 to 506 ms to initiate folding. The samples were then immediately pulse labeled with H₂O at pH 10.1 for 11 ms to rapidly label unstructured amides, followed by immediate quenching of the sample. The bimodal profiles during the folding process can be used to track the folding kinetics. The top and bottom panels are the controls for pulse labeling of the fully unfolded and folded states, respectively. Reproduced with permission from ref (380). Copyright 1997 American Chemical Society.

Figure 33

Pulse labeling HDX-MS used to track the aggregation of calcitonin. Each spectrum shows the deuteration profile (after a 2 min pulse of D₂O) starting from initial conditions (0 min) to the final time point at 1440 min. Populations I and II are clearly resolved and shift as the aggregates form over time. Reproduced with permission from ref (394). Copyright 2021 Elsevier.

Figure 34

Schematic representation of the continuum model of protein structure. The color gradient represents a continuum of conformational states ranging from highly dynamic, expanded conformational ensembles (red) to compact, dynamically restricted, fully folded globular states (blue). Dynamically disordered states are represented by heavy lines, stably folded structures as cartoons. A characteristic of IDPs is that they rapidly interconvert between multiple states in the dynamic conformational ensemble. In the continuum model, the proteome would populate the entire spectrum of dynamics, disorder, and folded structure depicted. Reproduced with permission from ref (403). Copyright 2014 American Chemical Society.

Figure 35

HDX-MS to measure residual structure in peptides. The exchange of two peptides compared to the predicted exchange rate is shown in (A) and (B). The predicted rates from k_ch are shown in dashed lines. (C,D) Exchange rates for predigested peptides (open circles) and peptide from intact protein (triangles) are compared directly to assess transient structure in the protein. Reproduced with permission from ref (66). Copyright 2017 American Chemical Society.

Figure 36

Deuterium uptake plots are shown for each peptide of unliganded HvASR1 (A), or bound to TFE (B), ZnSO₄ (C), or both TFE and ZnSO₄ (D). Points represented different time points of exchange denoted in the top of the figure. Figure adapted from ref (451).

Figure 37

HDX-MS comparison of xylose transporter in nanodiscs with different lipid compositions. The lower depiction shows the transmembrane helices in relation to the two membrane leaflets (gray). Differences are color coded onto the crystal structure. Figure adapted from ref (468).

Figure 38

HDX-MS of glycoproteins employing a postquench deglycosylation step. Glycoproteins are deuterium labeled in their fully native glycosylated state and after the quench step the glycopeptides generated through proteolysis are further digested using an acid-active endoglycosidase to remove the glycan and simply the LC-MS analyses of the deglycosylated peptides. Reproduced with permission from ref (520). Copyright 2020 American Chemical Society.

Figure 39

Difference in deuterium uptake for various glycoforms of trastuzumab (IgG) relative to reference material that is predominantly G0F with or without a single galactose (yellow circle, “G1F”). The structural effect of the glycosylation most readily evident in the Cγ2 domain. Figure adapted from ref (532).

Figure 40

Changes observed in the SARS-CoV-2 spike glycoprotein upon receptor binding. Beyond the stabilization seen at the receptor binding domain, there are also large changes observed within the furin cleavage site (672–690) and the stalk region (1175–1188). The long-range changes implicate the spike shifting to a primed state upon receptor binding, which leads to the activation of the protein to initiate membrane fusion. Figure adapted from ref (558).

Figure 41

Structural changes in phytochrome-activated guanylate cyclase upon red light illumination detected by HDX-MS. Regions becoming more accessible or more protected relative to the inactive (nonilluminated state) are shown in red and blue notes, respectively. Figure adapted ref (605).

Figure 42

Overview of solid-state HDX-MS. Lyophilized proteins are incubated with D₂O vapors to initiate exchange (top). Samples are then resuspended in quench buffer and processed by conventional HDX-MS approaches to measure deuterium uptake kinetics. Deconvoluted mass spectra for myoglobin are shown for the undeuterated (dotted line), dried with trehalose (solid line), and dried with sorbitol (dashed line) and the corresponding kinetic uptake curves (bottom). Figure is adapted from with permission from ref (697). Copyright 2015 MyJove.

Figure 43

Overview of histidine C₂ HDX-MS. The C₂ proton can undergo exchange through the formation of a ylide intermediate. Reproduced with permission from ref (127). Copyright 2008 American Chemical Society.

Figure 44

Various peptides from an interlab comparative HDX-MS study that controlled sample and labeling/quench buffers. Results for the same peptide from nine different laboratories are plotted to illustrate the interlab variability in the study. Reproduced with permission from ref (631). Copyright 2019 American Chemical Society.

Figure 45

Light and heavy (¹³C) caffeine standards used to measure variations in percent deuterium. The ratio of the heavy and light caffeine that were spiked into the protein solution and deuterium labeling solution accurately report the percentage of deuterium during the labeling step and account for solution dispending variability. Reproduced with permission from ref (632). Copyright 2014 American Chemical Society.

Figure 46

Deuterium uptake kinetics for the tripeptide YPI are shown in the presence of various buffer additives. The offset in the exchange profiles can be used to correct for the offset to the k_ch in the different solution, thereby enabling robust comparisons for proteins in the various solutions. Reproduced with permission from ref (131). Copyright 2017 American Chemical Society.

Figure 47

HX-PIPE combines both identification of clean features from the MS1 scans and peptide assignment from MS/MS data to accomplish peptide curation with less need for manual intervention. Reproduced with permission from ref (651). Copyright 2021 American Chemical Society.

Figure 48

Spectra are shown for an undeuterated (left) and deuterium labeled (right) peptide at either a mass resolution of 12 000 (a) or 750 000 (b). At ultrahigh resolution, the isotopomers within each apparent isotopic peak become resolved (c). Figure adapted from ref (655).

Figure 49

Deconvolution of multiple isotopic distributions using deMix. Isotopic distributions for three deuterium exchange time points are shown. The distributions could not be fit to a single distribution and thus were fit using two species (red and blue dashed lines). The w term in the inset shows the relative intensities of each species used in the fits. Figure adapted from ref (41).

Figure 50

HR-HDXMS uses a combination of overlapping peptides broken down into subfragments to estimate the thermodynamic properties with high spatial resolution, revealing a predicted stability fingerprint across the protein sequence. Figure adapted from ref (668).

Figure 51

Heat maps show deuterium uptake represented by different colors for each peptide across the primary sequence of the protein. Each bar shows the data for different time points, and the upper and lower set of bars represent two conditions of the protein. Reproduced with permission from ref (672). Copyright 2011 Elsevier.

Figure 52

Butterfly plots are used to compare two HDX experimental data sets. (A) The fraction deuterium uptake is plotted for all time points for all observable peptides for one protein sample (positive y-axis) and mirrored with another (negative y-axis). (B) The corresponding difference plot shows the subtracted result of the two data sets across all peptides and time points, revealing where changes are occurring. Reproduced with permission from ref (577). Copyright 2011 Elsevier.

Figure 53

HDX-MS studies of the transporter XylE. Effects of mutation on the exchange of XylE are plotted on the crystal structure (left). Woods plots show differences in the exchange between WT and mutant for all peptides (right). Regions becoming destabilized (red) and stabilized (blue) in the mutant are plotted according to their difference in deuterium uptake. Figure adapted from ref (478).

Figure 54

HDX-MS comparisons of Escherichia coli DinB polymerase represented by Chiclet plots. Differences are shown between free polymerase (E), the pol-DNA complex (ED), and the ternary pol-DNA-dNTP complex (EDN) with either the correct or incorrect dNTP. Comparisons for each peptide at each time point across different comparative sets are shown in the plots with coloring from more exposed (red) to no difference (gray) to more protected (blue). Areas with missing data are shown in white. Figure adapted from Nevin et al.

Figure 55

Examples of a volcano plots generated using Deuteros 2.0. The difference between two states of a protein are shown for all peptides and each time point. The x and y axis show the differences in deuterium uptake (ΔDU) and the p value, respectively. Quadrants that reflect statistically significant differences in exchange are shown in blue and red. Figure adapted from ref (680).