HDX-MS Technology Comparison: Systems, Case Studies, and Service Selection

Comparison of Mainstream HDX-MS Systems

The rapid development of hydrogen deuterium exchange mass spectrometry (HDX-MS) technology provides a powerful tool for the study of protein dynamic conformation and interaction, and plays a key role in drug development, structural biology and disease mechanism research.

However, different commercial HDX-MS systems (such as Thermo Fisher and Waters platforms) have significant differences in instrument design, automation and temperature control accuracy, which directly affect experimental efficiency and data reliability. At the same time, the application of this technology in the fields of antibody epitope analysis, neurodegenerative disease-related protein research and membrane protein complex structure analysis further highlights its multi-dimensional value.

In the face of the increasing demand for HDX-MS services, how to choose the appropriate technical solution based on the key indicators such as data reproducibility, customized analysis ability and software compatibility has become an urgent problem for researchers. Further research in this area will help optimize experimental design and promote more accurate protein dynamics studies.

Instrumentation Differences: Thermo Fisher vs. Waters

Core Platforms

• Thermo Fisher:

Typically employs Orbitrap-based systems (e.g., Q Exactive™ HF-X, Orbitrap Eclipse) coupled with UltiMate 3000 LC systems.

Detection: High-resolution mass analyzers (Orbitrap) provide <1 ppm mass accuracy and resolving power up to 500,000 (at m/z 200).

HDX Workflow: Uses LEAP PAL autosamplers with integrated cooling for deuterium exchange and quench steps.

• Waters:

Typically employs quadrupole-time-of-flight (QTof)-based systems (e.g., Xevo™ G2-XS, SYNAPT™ XS) coupled with ACQUITY™ UPLC™ I-Class systems.

Detection: High-resolution mass analyzers (QTof) provide <1 ppm mass accuracy (external calibration) and resolving power up to 60,000 (FWHM).

HDX Workflow: Uses Waters HDX Manager™ with integrated temperature control (0–40°C) and automated deuterium exchange/quench steps.

Automation & Workflow Integration

• Thermo Fisher:

Pros: Open-source software (e.g., HDExaminer, Chronos) allows third-party integration.

Cons: Requires manual optimization for quench conditions and LC gradients.

• Waters:

Pros: Tight integration with UNIFI and PLGS software; one-click HDX method setup.

Cons: Less flexibility for non-standard experiments (e.g., covalent labeling coupling).

Temperature Control

• Thermo Fisher:

Precision: ±0.1°C (via LEAP PAL cooling), but sample transfer lines may introduce variability.

Impact: Higher risk of back-exchange in long runs due to less standardized tubing insulation.

• Waters:

Precision: ±0.1°C (patented Peltier cooling in HDX manager).

Impact: Reduced back-exchange with fully enclosed, temperature-controlled fluidics.

Table 1. Comparison of parameters of Thermo Fisher and Waters HDX-MS systems

Automation Levels and Workflow Integration

Automation is a critical factor in HDX-MS workflows, impacting throughput, reproducibility, and ease of use. T1ermo Fisher and Waters systems take different approaches to automation, with trade-offs between flexibility and standardization.

Thermo Fisher HDX-MS Workflow Automation

• Sample Handling & Liquid Handling

LEAP PAL Autosampler:

Provides semi-automated deuterium labeling, quenching, and digestion.

Requires manual setup for cooling zones and reaction timing.

Pros: Flexible for custom protocols (e.g., variable labeling times, alternative quench buffers).

Cons: Less integrated; users must optimize fluidics to minimize back-exchange.

• LC-MS Integration

Vanquish/UHPLC + Orbitrap:

LC separation is controlled via Chromeleon or Xcalibur, while MS data is acquired in HDX mode.

Manual steps: Gradient optimization, column switching, and post-run data consolidation.

• Software & Data Processing

HDExaminer, Mass Spec Studio, or DynamX:

Third-party software requires manual peptide mapping and deuterium uptake calculations.

Pros: Highly customizable for differential HDX and time-resolved studies.

Cons: No native integration with Thermo's instrument software; prone to user bias in peak picking.

Waters HDX-MS Workflow Automation

• Sample Handling & Liquid Handling

HDX Manager Module:

Fully automated labeling, quenching, digestion, and injection.

Peltier-cooled flow path minimizes back-exchange.

Pros: Standardized protocols reduce variability; supports 96-well plates for high throughput.

Cons: Less adaptable to non-standard experiments (e.g., alternative proteases, kinetic studies).

• LC-MS Integration

ACQUITY UPLC M-Class + Synapt/Cyclic IMS:

UNIFI Software controls both LC and MS in a single interface.

Predefined HDX methods simplify setup but limit customization.

• Software & Data Processing

PLGS + DynamX (Waters):

Automated peptide identification (from MSE data) and deuterium uptake analysis.

Pros: Tight integration reduces manual steps; good for large-scale studies.

Cons: Less flexible for advanced analyses (e.g., hydrogen scrambling correction).

Temperature Control Precision and Impact on Data Quality

Temperature control is a critical factor in HDX-MS experiments, as even minor fluctuations can lead to deuterium back-exchange, reducing data accuracy and reproducibility. Both Thermo Fisher and Waters systems employ different strategies for temperature regulation, with direct consequences for peptide coverage, deuterium retention, and measurement reliability.

Table 2. Thermo Fisher vs. Waters system integration evaluation

Industry Application Case Studies

High-throughput HDX-MS has become indispensable in biopharmaceutical and structural biology research. Below are three key industry case studies highlighting how HDX-MS is applied in real-world scenarios, from biosimilar development to neurodegenerative disease research and membrane protein analysis.

Antibody Epitope Analysis for Biosimilar Development

Vascular endothelial growth factor A is required in both normal and pathological angiogenesis, as well as in embryonic angiogenesis. Although there are several other regulators of angiogenesis, the loss of the VEGF allele has been found to cause abnormal blood vessel growth and often death in embryos.

VEGF mRNA is overexpressed in most human tumors, and there is a link between mRNA expression levels and the vascular distribution of tumors.

Humanized bevacizumab (Avastin) is an IgG1 monoclonal antibody (mAb) developed as a cancer treatment. It binds to and neutralizes VEGF, preventing tumor angiogenesis by blocking receptor binding.

Time resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry (TRESI-HDX-MS) was used to detect subtle changes in the dynamics caused by the complexation. This method can obtain segmental average images of the dynamic changes of VEGF at the binding interface and complexation. Because HDX-MS is fast and reproducible, the method provides a reliable tool for screening potential biosimilar builds at an early stage of development.

Figure 1. Schematic illustration of a microfluidic device for time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Airtight syringes are used to transport solutions through capillary tubes. The antibody or antibody-antigen complex is transported through the inner capillary, while D2O flows through the outer capillary of the time-resolved kinetic mixer where the HDX reaction occurs. Valco Mixing T is used to connect a kinetic mixer to a capillary tube transporting acetic acid to allow mixing and subsequent reaction quenching. The solution is then transported to the protease chamber, where it is digested before entering the mass spectrometer via a metal capillary tube used as an ESI probe. (Brown KA et al., 2020)

Protein Dynamics in Neurodegenerative Diseases: TDP-43 Aggregation

The formation of insoluble cytoplasmic aggregates of the RNA-binding protein TDP-43 is a major hallmark of neurodegenerative diseases, including amyotrophic lateral sclerosis.

TDP-43 is localized primarily in the nucleus and arranges itself into dynamic condensates by liquid-liquid phase separation (LLPS). Mutations and post-translational modifications can alter the condensation properties of TDP-43, facilitating the transformation of liquid biomolecular agglomerates into solid aggregates.

However, studying the dynamics of this process in vivo has been a challenge to date. Here, Minshull TC et al. address this challenge using hydrogen–deuterium exchange-mass spectrometry.

Minshull TC et al. compared the degree of deuterium incorporation after we compared a rapid labeling pulse (30 seconds) with a fully deuterium-labeled sample, and obtained the results of conformational dynamics changes by analysis.

Figure 2. HDX-MS reveals dynamic disorder in monomeric TDP-43 and salt-dependent changes in conformational dynamics. (Minshull TC et al., 2024)

Membrane Protein Complex Structure Analysis Using Native MS-Coupled Approaches

Protein folding is a complex and precise process in living cells. Most of the exported proteins escape cytoplasmic folding, target the membrane, and then transport to/across the membrane. Their targeting and translocation capability states are non-locally folded. However, once they reach the appropriate cell compartment, they can fold back into their native state.

The unnatural state of the preprotein remains structurally poor, as increased disorder, protein size, tendency to aggregate, and time scales for observation are often limiting factors for typical structural methods such as X-ray crystallography and NMR.

Using HDX-MS, A method based on differentiated isotope exchange rates in structured versus unstructured protein states/regions, and highly dynamic versus more rigid regions, Tsirigotaki A. et al. propose a complete pipeline of structural characterization, from peptide preparation to data analysis and interpretation.

Service Selection Criteria for HDX-MS

When outsourcing Hydrogen-Deuterium Exchange Mass Spectrometry to Contract Research Organizations or core facilities, selecting the right provider requires careful evaluation of data quality, analysis capabilities, and workflow compatibility. Below are the key criteria to consider

Data Reproducibility and Sequence Coverage Benchmarks

Critical Metrics:

• Sequence Coverage (>90% for soluble proteins, >70% for membrane proteins)
• Peptide Redundancy (≥3 peptides per region for reliable deuteration analysis)
• Back-Exchange Control (<10% for high-quality data)
• Inter-Lab Reproducibility (CV < 10% for deuterium uptake measurements)

How to Evaluate:

• Request sample datasets from past projects (e.g., antibody-antigen complexes).
• Check if the provider follows best practices (e.g., USP <1085> guidelines for HDX-MS).
• Ask about QC steps (e.g., internal standards, replicate runs).

Human Metabolome Database (HMDB): The HMDB offers extensive data on human metabolites, including biochemical properties, tissue concentrations, and disease associations. It facilitates spectral matching, helping researchers identify metabolites in experimental data and retrieve information on their biological roles. The HMDB is also instrumental in biomarker discovery by providing reference data on metabolite levels under different physiological conditions.

Kyoto Encyclopedia of Genes and Genomes (KEGG): KEGG integrates data from various biological disciplines, including metabolomics, and provides detailed information on metabolic pathways, gene functions, and biochemical reactions. Researchers use KEGG for pathway mapping, which helps visualize how metabolites interact within metabolic networks. This resource is essential for understanding the impact of metabolic changes on cellular processes and identifying potential therapeutic targets.

MetaCyc and BioCyc: MetaCyc offers a curated collection of metabolic pathways for a wide range of organisms, while BioCyc provides detailed pathway information for specific species. These databases are used to reconstruct and analyze metabolic networks, helping researchers map metabolite data onto these networks to identify altered pathways or key regulatory nodes in response to biological perturbations.

Custom Analysis Capabilities: Time-Resolved and Differential HDX-MS

Table 3. Custom options for user reference

Software and Data Analysis Tools: Automation and Flexibility

Table 4. Software parameter comparison