The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.
Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson’s disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.
Parkinson's disease (PD) is a neurodegenerative illness affecting millions of people worldwide1. It is characterized by the formation of cytoplasmic inclusions known as Lewy bodies and Lewy neurites in the brain's substantia nigra pars compacta region. These cytoplasmic inclusions have been found to contain aggregates of the intrinsically disordered protein aSyn2. In PD and other synucleinopathies, aSyn transforms from a soluble disordered state into an insoluble, highly structured diseased state. In its native form, monomeric aSyn adopts a wide range of conformations stabilized by long-range electrostatic interactions between its N- and C-termini and hydrophobic interactions between its C-terminus and non-amyloid beta component (NAC) region3,4,5,6. Any disruptions in those stabilizing interactions, such as mutations, post-translational modifications, and changes in the local environment, can lead to the misfolding of the monomer, thus triggering the process of aggregation7.
While a vast amount of research exists on the oligomeric and fibrillar forms of aSyn8,9,10,11, there is a crucial need to study the monomeric form of the protein and better understand which conformers are functional (and how) and which are prone to aggregate8,9,10,11. Being intrinsically disordered, only 14 kDa in size, and difficult to crystallize, the aSyn monomer is not amenable to most structural biological techniques. However, one technique capable of measuring the conformational dynamics of monomeric aSyn is millisecond HDX-MS, which has recently generated important structural observations that would be challenging or impossible to obtain otherwise12,13,14. Millisecond HDX-MS sensitively measures the average of the protein conformational ensemble by monitoring the isotopic exchange at amide hydrogens, indicating solvent accessibility and hydrogen-bonding network participation of a particular protein region on the millisecond timescale. It is necessary to stress the millisecond aspect of the HDX-MS as, due to its natively unfolded, meta-stable nature, aSyn exhibits very fast hydrogen-exchange kinetics that manifest well below the lower limit of conventional HDX-MS systems. For example, most of the aSyn molecule has completely exchanged hydrogen for deuterium under intracellular conditions in less than 1 s. Several laboratories have now built fast-mixing instrumentation; in this case, a prototype fast-mixing quench-flow instrument capable of performing HDX-MS with a dead-time of 50 ms and a temporal resolution of 1 ms is used15. While millisecond HDX-MS has recently been acutely important in the study of aSyn, it stands to be valuable in studying intrinsically disordered proteins/regions more widely and a large number of proteins with loops/regions that are only weakly stable. For example, peptide drugs (e.g., insulin; GLP-1/glucagon; tirzepatide) and peptide-fusion proteins (e.g., the HIV inhibitor FN3-L35-T1144) are major drug formats where solution-phase structural and stability information can be a critical input for drug development decisions, and, yet, the peptide moiety is often only weakly stable and intractable by HDX-MS at the seconds timescale16,17,18,19,20. Emergent HDX-MS methods with labeling in the seconds/minutes domains have been shown to derive structural information for DNA G-quadruplexes, but it should be possible to extend this to more diverse oligonucleotide structures by the application of millisecond HDX-MS21.
HDX-MS experiments can be performed at three different levels: (1) bottom-up (whereby the labeled protein is digested proteolytically), (2) middle-down (whereby the labeled protein is digested proteolytically, and the resulting peptides are fragmented further by soft-fragmentation techniques), and (3) top-down (whereby soft-fragmentation techniques directly fragment the labeled protein)22. Thus, sub-molecular HDX-MS data allow us to localize the exchange behavior to specific regions of a protein, making it critical to have adequate sequence coverage for such experiments. The structural resolution of any HDX-MS experiment relies on the number of proteolytic peptides or fragments derived from the protein upon digestion or soft-fragmentation, respectively. In each of the three experiment types outlined above, the change in amide exchange at each peptide/fragment is mapped back onto the protein's primary structure to indicate the behavior of localized regions of the protein. While the highest structural resolution is achieved through soft-fragmentation, the description of these experiments is out of the scope of the current study, which focuses on the measurement of aSyn monomer conformations. Excellent results can be obtained with the commonly applied "bottom-up" workflow described here.
Here, procedures are provided on (1) how to prepare and handle aSyn samples and HDX-MS buffers, (2) how to perform peptide mapping for a bottom-up HDX-MS experiment, (3) how to acquire HDX-MS data on monomeric aSyn under physiological conditions, specifically in the millisecond time domain (using a custom-built instrument; alternative instruments for millisecond labeling have also been described), and (4) how to process and analyze the HDX-MS data. Methods using monomeric aSyn at physiological pH (7.40) in two solution conditions are exemplified here. While critically useful in the study of aSyn, these procedures can be applied to any protein and are not limited to intrinsically disordered proteins.
1. Protein expression and purification of aSyn
2. HDX buffer preparation
NOTE: Since Tris has a high temperature coefficient, the pH measurement needs to be adjusted for the temperature at which the HDX reaction will be done, which is 20 °C in this protocol.
3. Peptide mapping procedure
4. Millisecond hydrogen/deuterium exchange study
5. Data processing
6. Data analysis
Due to its intrinsically disordered nature, it is difficult to capture the intricate structural changes in aSyn at physiological pH. HDX-MS monitors isotopic exchange at backbone amide hydrogens, probing the protein conformational dynamics and interactions. It is one of the few techniques to acquire this information at high structural and temporal resolutions. This protocol is broadly applicable to a wide range of proteins and buffer conditions, and this is exemplified by the measurement of the exchange kinetics of aSyn in two different solution conditions: State A and State B8, as defined in steps 2.1.-2.2.
First, a mapping experiment on aSyn was performed, and a peptide coverage map was obtained, as shown in Figure 1. The map covers 100% of the protein sequence and has an average redundancy of 3.79. The 100% coverage value indicates that all the amino acids in the protein were found in the protein digests and will enable a comprehensive analysis of the exchange behavior of aSyn. The redundancy value indicates the number of overlapping peptides. A higher redundancy value increases the structural resolution of the final map, given subtractive flattening of the data for overlapping peptides32.
Using the fast-mixing quench-flow instrument prototype (see Table of Materials), high-quality, millisecond-timescale HDX-MS data on aSyn at pH 7.4 in State A and State B were collected (Figure 2). Following an isotopic assignment in DynamX, "crude" deuterium uptake curves were obtained, as shown in Figure 3A. It shows uptake curves for three peptides selected across each protein domain. The deuterium incorporation over time is displayed. The x-axis is on the millisecond timescale, which aligns with the very fast kinetics of aSyn at physiological conditions. The red shaded region shows the data typically obtained from conventional HDX instruments, with starting measurements from 30 s. Importantly, this cannot be further reduced by pH manipulation for so-called "time-window expansion"; that approach is invalid for the study of intrinsically disordered proteins/regions, as the pH shift will perturb the conformational ensemble of the weakly stable polypeptide. As can be seen here, most of aSyn is fully exchanged by 1 s (Figure 3C). This shows the importance of millisecond HDX measurements for monomeric aSyn as the full kinetic uptake curve for the exchange reaction is captured, which yields the most accurate measurement of the monomer conformations.
HDfleX performed back-exchange correction using the plateau deuterium incorporation. The data points were subsequently fitted according to Equation 1, providing an observed rate constant, kobs, indicative of the solvent accessibility and hydrogen bonding involvement of that particular peptide (Figure 3B).
Equation 1
where Dt is the deuterium incorporation at time t, nExp is the number of exponential phases, N is the maximum number of labile hydrogens, kobs is the observed exchange rate constant, and β is a stretching factor30,33.
Following curve fitting, the uptake area under the fitted curve can be calculated by integrating the fitted function describing the uptake curve within the experimental time window. Statistical significance analyses between the uptake area of the two states were performed. First, a global significance threshold for the uptake area was calculated in HDfleX at a confidence level of 95%. Uptake area difference plots were then generated, showing the difference between State A and State B at two levels of structural resolution: peptide resolution (Figure 4A) and amino acid resolution (Figure 4B). The peptide resolution difference plot shows the difference in uptake area between State A and State B for each individual peptide, while the amino acid resolution difference plot shows the difference in uptake area between State A and State B flattened across the entire amino acid sequence of aSyn30,34. Both plots indicate an overall greater deuterium uptake throughout the aSyn monomer in State B compared to State A. This finding can be justified by examining the deuterium uptake plots in Figure 3, where the State B uptake curve is always above the State A uptake curve. Furthermore, it can be seen that the magnitude of the uptake area difference is much higher at the C-terminus. Once again, this can be justified by tracing back to the original uptake curves, where the C-terminal peptides (peptides 124-140 shown in Figure 3) show a much bigger gap between the uptake curves than the rest of the protein. In conclusion, the solution conditions in State B cause an increase in solvent exposure or decrease in hydrogen-bonding network participation throughout the protein but more so at the C-terminus.
Figure 1: Peptide coverage map of wild-type aSyn with a total of 30 peptides and 100% sequence coverage. The three domains of aSyn are highlighted as follows: N-terminus (blue), non-amyloid beta component region (yellow), and C-terminus (red). Please click here to view a larger version of this figure.
Figure 2: Workflow for a millisecond HDX-MS experiment on aSyn. Please click here to view a larger version of this figure.
Figure 3: Example uptake plots from three peptides selected across the three domains of aSyn for State A (yellow) and State B (blue). (A) Unfitted and non-back-exchange corrected uptake plot. (B) Fitted and back-exchange corrected uptake plots. The red shaded region represents data obtainable by conventional HDX-MS systems, typically starting from 30 s. Error bars correspond to the standard deviation of the three replicates. (C) Heatmap plot of the percentage deuterium uptake across amino acid sequence per timepoint. The color bar represents the percentage of deuterium uptake. Please click here to view a larger version of this figure.
Figure 4: Uptake area difference plots at maximum curve plateau time (14,084 ms). (A) Peptide residue resolution plots show each peptide's uptake area difference between State A and State B. (B) Amino acid resolution plot showing the uptake area difference between State A and State B flattened across the amino acid sequence. Please click here to view a larger version of this figure.
Mapping Energy Level | Ramp Voltage (V) |
Low | 20-40 |
Medium | 25-45 |
High | 30-50 |
Very high | 35-55 |
Table 1: Mapping energy levels and corresponding transfer ramp voltages.
In the present article, the following procedures are described: (1) performing peptide mapping experiments on monomeric aSyn to obtain the highest sequence coverage, (2) acquiring millisecond HDX-MS data on monomeric aSyn under physiological conditions, and (3) performing data analysis and interpretation of the resulting HDX-MS data. The provided procedures are generally simple to execute, each labeling experiment typically lasts only around 8 h for three replicates and eight timepoints, and the mapping experiment lasts only around 2 h. Given the fully-automated instrumentation used here, a complete dataset can be acquired in 1 day. However, when handling samples and preparing buffers, care must be taken to ensure that measurements are derived from weakly stable protein (or protein regions) in the desired state. Importantly, a previous study showed that different storage conditions, such as freezing and lyophilizing, resulted in different aSyn monomer conformers and that it is important to characterize the potential impact of sample handling on the aSyn monomer conformational ensemble10. Indeed, HDX-MS is a highly sensitive measure of such conformational perturbations, with a dynamic range from microseconds to at least months. In addition, if strictly studying only the aSyn monomer, filtration is strongly advised to remove unwanted oligomers and fibrils that might have formed in the sample upon storage or handling. Furthermore, the HDX-MS buffers need to be tightly controlled within 0.05 of the desired pH or pD, as any discrepancies will significantly affect the intrinsic rate of exchange and lead to unwanted errors. It is also important to note that comparisons between solution conditions for any protein that differs in pH, temperature, or salt composition will alter the intrinsic rate. Therefore, these data will require further corrections, such as applying a pH adjustment factor35 or an empirical correction factor8,30.
In terms of instrumentation, there are no commercially available systems that allow the acquisition of millisecond HDX-MS data. Several research groups have developed their own systems, from quench-flow systems13,15,36 to microfluidic chips37,38,39 to capture the rapid exchange kinetics of certain proteins. Another method that has been used to achieve millisecond timescale HDX-MS data is known as the time expansion method40,41, whereby the pH of the buffers is reduced to slow down the exchange kinetics. However, this method does not apply to aSyn (or to any weakly stable protein features) as (1) the lowering of the pH drastically changes the charge density of the protein and increases the rate of aggregation8,42, and (2) the aSyn conformers are only meta-stable and are likely to be perturbed by these pH alterations. For these reasons, it is recommended to maintain a consistent pH in the HDX-MS buffers when studying monomeric aSyn conformations, unless physiologically relevant, and employ a millisecond labeling instrument.
Most of the aSyn monomer fully exchanges within 1 s, and at most, it takes approximately 15 s to fully exchange with deuterium (Figure 3) at a physiologically relevant pH of 7.4 (reflective of intracellular cytosolic conditions at the presynapse). Using conventional HDX-MS systems, starting at 30 s is not appropriate as the HDX-MS data would correspond to the plateau of the exchange reaction, which does not provide any useful conformational information. However, the lower limit of measurement of the millisecond HDX instrument (corresponding to a "dead-time" of 50 ms) enables monitoring of the exchange reaction from ~25% completion for the aSyn monomer at pH 7.4. This allowed us to capture the majority of the kinetic uptake curve. Fitting the deuterium uptake curve to Equation 1 provides important kinetic information; it corresponds to an estimate of the observed rate constant, kobs. While not covered here, it is possible to carry out aggregation kinetics experiments and examine fibril morphologies of aSyn under the same solution conditions as the HDX-MS experiments since HDX-MS is highly tolerant of a wide range of buffers8. Thus, for example, the kobs from the HDX-MS experiment can be correlated with the results from the aggregation experiments to gain insight into which conformations are most prone to certain aggregation behaviors and fibril morphologies.
For the simple case of differential HDX-MS experiments, where two or more conditions or protein variants are to be compared, the area under the fitted uptake curve can be integrated for each state and compared to each other. In this study, the uptake areas for State A and State B were compared at two different levels of structural resolution: peptide resolution and amino acid resolution, both of which have distinct strengths and challenges. For instance, the peptide resolution data reflect the raw spectral data more closely and have undergone the least processing. However, the "flattened" amino acid resolution data allow both peptide and soft-fragmentation information to be combined into a single output rather than separate unmergeable outputs and, ultimately, present the data at the highest structural resolution. One limitation of the mass spectrometry detection of HDX labeling is the challenge of obtaining amino acid resolution. While "soft-fragmentation" techniques, such as electron transfer dissociation (ETD), electron capture dissociation (ECD), and ultraviolet photodissociation (UVPD), have been proven to be effective at generating higher resolutions, they remain challenging, unpredictable, and inefficient30,43,44,45,46,47,48.
Compared to other structural techniques, millisecond HDX-MS has the unique advantage of capturing the conformational dynamics of monomeric aSyn at high structural and temporal resolutions. As the fast exchange kinetics of the monomer are no longer a limiting factor, further studies can be performed on monomeric aSyn with different mutations, post-translational modifications, salt components and concentrations, and binding partners at physiological pH. Correlating the HDX-MS results with functional studies, such as aggregation kinetics and fibril morphologies, can provide insight into conformers that either promote normal cellular function or are disease-prone. Ultimately, it is anticipated that such millisecond HDX-MS may be crucial for discovering targeted drugs that stabilize specific physiologically tolerated conformers.
The authors have nothing to disclose.
NS is funded by the University Council Diamond Jubilee Scholarship. JJP is supported by a UKRI Future Leaders Fellowship [Grant number: MR/T02223X/1].
1 × 100 mm ACQUITY BEH 1.7 μm C18 column | Waters Corporation | 186002346 | Analytical column |
Acetonitrile HPLC grade >99.9% HiPerSolv | VWR | 20060.420 | For LC mobile phases |
CaCl2 | Sigma Aldrich | C5670 | Salt for HDX buffers |
Chronos | Axel Semrau (Purchased from Waters Corporation) | 667006090 | Scheduling software to enable multiple HDX-MS sample injections automatically. Alternative software is available from other vendors e.g. HDXDirector or LEAP Shell |
Deuterium chloride | Goss Scientific (Cambridge Isotope Laboratories) | DLM-2-50 | For HDX labelling buffers |
Deuterium oxide (99.9% D2O) | Goss Scientific (Cambridge Isotope Laboratories) | DLM-4 | Deuterated water |
DynamX 3.0 | Waters Corporation | 176016027 | Isotopic assignment and deuterium incorporation calculation |
Enzymate BEH Pepsin Column | Waters Corporation | 186007233 | Pepsin digestion column |
Formic Acid, 99.0% LC/MS Grade | Fisher Scientific | 10596814 | For LC mobile phases |
Guanidinium hydrochloride | Sigma Aldrich | RDD001-500G | Chaotrope/Denaturant |
HDfleX | University of Exeter | N/A | https://ore.exeter.ac.uk/repository/handle/10871/127982 |
KCl | Sigma Aldrich | P3911 | Salt for HDX buffers |
LEAP HDX-2 CTC PAL sampling robot | Waters Corporation | 725000637 | Autosampler robot |
Leucine enkephalin | Waters Corporation | 186006013 | For mass spectrometry lockspray calibration. |
MassLynx | Waters Corporation | 667004007 | Software controlling inlet methods and mass spectrometer |
Maximum recovery vials | Waters Corporation | 600000670CV | 100 pack including caps – used for quench tray in LEAP HDX-2 |
MgCl2 | Sigma Aldrich | M8266 | Salt for HDX buffers |
Millipore 0.22 µm syringe filters | Millipore | N9CA7069B | Syringe filters |
ms2min | Applied Photophysics Ltd | N/A | fast-mix quench-flow millisecond hdx instrument |
NaCl | Sigma Aldrich | S9888 | Salt for HDX buffers |
Peltier temperature controller | LEAP Technologies Inc. | HP115-COOL/D | Peltier controller to set precise temperature of chambers in the LEAP robot. |
ProteinLynx Global Server 3.0 | Waters Corporation | 715001030 | Peptide identification software. Alternative software is available from other vendors. |
Reagent pot caps | Waters Corporation | 186004632 | 100 pack |
Reagent pots for LEAP HDX-2 | Waters Corporation | 186001420 | 100 pack excluding caps – used for buffers in LEAP HDX-2 |
Sodium deuteroxide (99.5% in D2O) | Goss Scientific (Cambridge Isotope Laboratories) | DLM-57 | For HDX labelling buffers |
Spin filter microcentrifuge tubes (3 kDa MWCO) | Amicon (Merck Sigma Aldrich) | UFC5003 | Micro centrifuge tubes to concentrate protein. This facilitates buffer exchange and accurate sample loading for HDX-MS experiments. |
Synapt G2-Si mass spectrometer | Waters Corporation | 176850035 | Mass spectrometer |
Total recovery vials | Waters Corporation | 600000671CV | 100 pack including caps – used for labelling tray in LEAP HDX-2 |
Tris-HCl | Sigma Aldrich | T3253-250G | Buffer |
Trizma base | Sigma Aldrich | T60040-B2005 | Buffer |
Urea | Sigma Aldrich | U5378-1KG | Chaotrope/Denaturant |
VanGuard 2.1 x 5 mm ACQUITY BEH C18 column | Waters Corporation | 186004623 | Trap desalting column |