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. 2026 Mar 9;42(4):btag113. doi: 10.1093/bioinformatics/btag113

HXMS: a standardized file format for HX-MS data

Kyle C Weber 1,†, Chenlin Lu 2,†, Roberto Vera Alvarez 3, Bruce D Pascal 4, Anum Glasgow 5,✉
Editor: Lenore Cowen
PMCID: PMC13075950  PMID: 41803064

Abstract

Motivation

Hydrogen/deuterium exchange-mass spectrometry (HX-MS) is a rapidly expanding technique used to investigate protein conformational ensembles. The growing popularity and utility of HX-MS has driven the development of diverse instrumentation and software, resulting in inconsistent, non-standardized data analysis and representation. Most HX-MS data formats also employ only mean deuteration representations of the data rather than full isotopic mass spectra, which reduces the information content of the data and limits downstream quantitative analysis.

Results

Inspired by reliable protein structure and genomics data formats, we present HXMS, a unified, lightweight, scalable, and human-readable file format for HX-MS data. The HXMS format preserves the isotopic mass envelopes for all peptides, captures the full experimental time-course including fully deuterated control samples, and contains all other key information. It supports multimodal distributions, post-translational modifications (PTMs), and experimental replicates. To promote compatibility with existing HX-MS workflows, we also developed PFLink, a Python package that converts exported data files from commonly used HX-MS software to the HXMS format. PFLink and the HXMS format will enable quantitative, higher-resolution data processing, improved data sharing and storage among HX-MS practitioners, future machine learning applications, and further developments in HX-MS analysis.

Availability and implementation

PFLink is publicly available to install locally on HuggingFace, alongside documentation, or use online at HuggingFace (https://huggingface.co/spaces/glasgow-lab/PFlink). The supplementary information includes sample input files, sample HXMS files, and a generic unfilled PFlink custom CSV file that users may populate with key experimental conditions and results, which can then be read and converted into the HXMS format.

1. Introduction

Hydrogen/deuterium exchange-mass spectrometry (HX-MS) is a powerful biophysical method for probing protein folding and conformational ensembles (Englander and Kallenbach 1983, Englander et al. 2016). HX-MS measures the rate at which backbone amide hydrogens in a protein are replaced with deuterium atoms when exposed to deuterated buffers, providing time-resolved insights into protein structure and conformational stability (Hamuro 2024). The availability of automated systems to perform HX-MS experiments (Chalmers et al. 2006, Wales et al. 2008, Wei et al. 2014, Kish et al. 2023) and analyze HX-MS data has increased its appeal for studying large, complex proteins, allowing scientists to probe many pharmacological targets for applications such as drug discovery, epitope mapping, and protein characterization (Gertsman et al. 2009, Wang et al. 2012, Hamuro 2017, Gramlich et al. 2021, Glasgow et al. 2023, Jia et al. 2023, Shaw et al. 2023, Wales et al. 2024, Wells et al. 2025).

Despite the widespread adoption of HX-MS in the structural biology community, most practitioners analyze data at the peptide level using the mean deuteration of the isotopic mass envelope at each time point. This quantity is often referred to as the “centroid” representation of the data, although this usage differs from the mass spectrometry convention of centroiding profile spectra into stick representations. Although it is commonplace, the practice of using MS mean deuteration rather than the full MS envelopes limits opportunities for quantitative treatment of HX-MS data due to the resulting information loss and degeneracy (Kan et al. 2019, Lu et al. 2026). This problem is exacerbated by the large size and instrumentation-specific formats of raw MS data files, and the varied export formats of available HX-MS software. Altogether, while there have been efforts to standardize reporting in the field (Masson et al. 2019), sharing HX-MS data remains difficult and cumbersome since no suitable standardized format exists.

Here we present HXMS, a lightweight and human-readable file format for HX-MS data that includes the experimental isotopic mass envelopes and all necessary information for high-resolution data analysis. We also introduce PFLink, a software package that converts HX-MS data files produced by four different commercial and academic software packages, or our custom data input file, to the HXMS format (Fig. 1). The standardized HXMS format will advance data processing, data sharing, and technical developments within and beyond the HX-MS community.

Figure 1.

For image description, please refer to the figure legend and surrounding text.

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HXMS file creation using PFLink. The user provides two types of information to PFLink: (i) HX-MS data exported directly from the analysis software, or in the PFLink custom format, and (ii) details about the protein and experimental conditions. PFLink automatically compiles this information in the standardized HXMS format.

2. Results

2.1. Metadata section format

The HXMS file format consists of three main sections: a metadata section, an experimental data section, and a post-translational modification (PTM) dictionary section. The metadata section describes the required experimental conditions used in the HX-MS experiment (Table 1). Important variables include the protein name, sequence, and state; the temperature, the pH(read), and the D2O saturation. The user may add additional metadata using a “REMARK” header in the metadata section. Metadata objects must start with either “METADATA” or “REMARK,” followed by a tab. The title comes next, followed by a final tab before the data or remark itself, and ends with a new line character. The HXMS file version is automatically added to the “REMARK” section. The current version is v1.0.

Table 1.

Metadata information and requirements.

Metadata Information Example Required
PROTEIN_SEQUENCE Protein sequence GSHMKTVEVNGADASDDN Yes
PROTEIN_NAME Name of the protein Human PFK-1 No
PROTEIN_STATE State of the protein APO No
TEMPERATURE (K) Temperature in K 293.15 Yes
pH(READ) pH(read) 6.0 Yes
D2O_SATURATION D2O saturation 0.91 Yes
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2.2. Experimental data section format

The experimental data section contains all information necessary to represent a singular timepoint in any HX-MS experiment (Table 2). In the HXMS file format, each field is represented as a column in the tabular data section. The “TP” tag is used to declare a timepoint, followed by a peptide timepoint index, “INDEX,” which incrementally increases by 1. If any timepoint represents a peptide that exhibits a multimodal distribution, the “MOD” column designates each distinct population using alphabetical indices (A–Z). A single, unimodal distribution is labeled “A” by default, whereas additional coexisting populations are assigned subsequent alphabetical indices (e.g. “B,” “C,” etc.) in the “MOD” column, thereby supporting extensible multimodal behavior. The “START” and “END” columns indicate the peptide’s beginning and end indices. These are inclusive of both ends, and indexing starts at 1 for the first amino acid in the protein. If there are multiple experimental measurements of the same peptide, the “REP” column may be indexed to denote this, where index 0 is the default for a single replicate. Replicates should be incremented only for new experimental measurements, not for timepoints in the same experiment. The “PTM_ID” column catalogs PTMs. For each unique PTM, whether it is on the same peptide or a different one, the counter is incremented. The default setting for no PTM uses the index 0000. The “PTM_ID” is used in the lookup table in the PTM section of the HXMS file to provide information about each modification, as described further in Section 2.3. The “TIME(SEC)” column indicates the duration of the sample incubation in D2O before it was quenched. Time is reported in scientific notation, except for fully deuterated samples, which are reported as “inf.” The “UPTAKE” column denotes the amount of deuterium incorporated. This is calculated by subtracting the mean deuteration of the 0 s timepoint from the mean deuteration for each given timepoint, for each peptide and replicate. Finally, the “ENVELOPE” column contains full isotopic mass envelopes when envelope-level spectral data are available. There is no limit to the number of peaks that can be included. The peaks are separated by commas and are normalized to sum to 1.

Table 2.

HX-MS data.

Header name Information Number of characters Separation character Example
TITLE Type of data 12 N/A TP
INDEX Peptide index 8 N/A 0
MOD Multimodal index 7 N/A A
START Peptide start position 7 N/A 1
END Peptide end position 7 N/A 10
REP Experiment number 5 N/A 0
PTM_ID PTM ID used for the PTM lookup table 8 N/A 0000
TIME(SEC) Incubation time in D2O 16 N/A 0.000000e+00
UPTAKE Mean deuteration uptake relative to 0 time point 9 N/A 0.00
ENVELOPE Mass spec full envelope inf, 4 per peak , 0.527,0.298,0.116,0.036,0.000,0.023,0.000
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To account for back exchange, the HXMS format includes fully deuterated controls as standard timepoints within the experimental data section. These entries follow the same structure as other timepoints and allow replicates, with “TIME(SEC)” explicitly set to “inf” to enable consistent quantification and correction. The UPTAKE column reports raw deuterium incorporation and is not corrected for back exchange.

2.3. PTM section format

The PTM section serves as a dictionary, providing detailed information for any PTMs in the dataset (Table 3). This section links the “PTM_ID” from the experimental data section to a comprehensive description of the modification. The “PTM” tag is used to declare a PTM, followed by the “PTM_ID” in the timepoint series section. The description of the entry is in the “CONTENT” column, which describes the modified amino acid within the protein sequence. If there are multiple PTMs in one peptide, one may place a comma and denote the next PTM on the same line. We recommend using PDB ligand ID for simplicity and clarity, but any declaration format can be used due to the high diversity of PTMs.

Table 3.

PTM format.

Header name Information Number of characters Separation character Example
TITLE Type of data 12 N/A PTM
PTM_ID PTM ID used for the PTM lookup table 8 N/A 0000
CONTENT The PTM denoted by the absolute position of the PTM in the peptide and PTM ID inf , Phosphoryl STY (18)
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Table 4.

MATCH format.

Header name Information Number of characters Separation character Example
TITLE_MATCH Type of data 12 N/A MATCH
TP_ID TP ID used for MATCH lookup 8 N/A 1
CONF Confidence score for the matched envelope assignment 8 N/A 1.00
RT(min) Chromatographic retention time, minutes 8 N/A 8.680
Z Charge state 5 N/A 4
MONO_M Monoisotopic mass of the matched peptide 16 N/A 2091.977598
M/Z Raw isotopic mass envelope data inf

  • , separates envelope peaks corresponding per TP row

  • ; separates fine isotopologue structure within each envelope peak

  • : separates m/z and intensity

 523.9764:2.076; 523.9792:2698.8441; 523.982:1595.0798,524.319:7.059; 524.3218:3170.0975; 524.3246:8130.5251; 524.3273:9330.6525; 524.3301:7816.5479; 524.3329:3718.9259; 524.3357:1818.192
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2.4. MATCH section format

The optional MATCH section is designed to preserve the raw matched isotopic envelope information associated with each timepoint “TP”. This section functions as a lookup table linking processed timepoint data to the original spectral evidence, thereby ensuring full traceability of peak assignments. The “MATCH” tag specifies the data type. Each entry is indexed by “TP_ID,” which corresponds to the identifier used in the timepoints. The “CONF” field records the confidence score for the matched envelope assignment. The “Z” denotes the charge state of the peptide ion. The MONO_M field reports the monoisotopic mass of the matched peptide.

The “M/Z” header stores the raw isotopic mass envelopes using a hierarchical delimiter format. Individual envelope peaks corresponding to entries in the timepoint table are separated by commas “,”. Within each envelope peak, uncentroided fine structures of the spectra, including isotopologues, are separated by semicolons “;”. For each peak or isotopologue, the m/z value and its intensity are separated by a colon “:”. This design ensures that the complete raw matched envelope information is retained in a structured and reproducible format.

2.5. PFLink: software to convert HX-MS files to the HXMS format

To enable widespread compatibility and accessibility with the HXMS format, we developed PFLink, a Python package to convert exported HX-MS data files from several widely used commercial and academic HX-MS data analysis programs. PFLink is compatible with exported data from BioPharma Finder (Thermo Fisher), HDExaminer (Trajan), DynamX (Waters), and HDX Workbench (Pascal et al. 2012) (Fig. 1, Supplementary Data 1). As most HX-MS analysis software packages currently only support the export of HX-MS data in the mean deuteration format, PFLink can write HXMS files originating from any of these programs in the mean deuteration format.

BioPharma Finder and DynamX report deuterium uptake directly, so HX-MS uptake data exported from these programs can be used as-is. In contrast, PFLink recalculates deuterium uptake as reported by HDX Workbench by first identifying the zero timepoint “centroid” value for each peptide (or an average of the replicates, if any replicate is missing a zero timepoint), and then normalizes each timepoint “centroid” value by subtracting this zero timepoint value. PTMs are supported on all file formats except for HDExaminer and BioPharma Finder; for these programs, they must be added to the HXMS file manually.

Because both HDX Workbench and HDExaminer support exporting complete isotopic mass spectra for all peptides, PFLink can also generate HXMS files in the full-spectrum format when provided with this input. Leveraging the full isotopic mass spectra exported by HDX Workbench and HDExaminer, PFLink can generate the MATCH section, preserving either centroided envelopes or uncentroided isotopologue fine structure as structured m/z-intensity pairs, thereby maintaining direct traceability to the underlying raw spectral data. Alternatively, users may choose to run PFLink using the custom data format supplied in Supplementary Data 1. The resulting HXMS files are compatible with the quantitative and high-resolution HX-MS analysis methods PFNet (Lu et al. 2025) and FEATHER (Lu et al. 2026).

2.6. Two examples

We include two sets of HXMS files on two proteins as examples (Supplementary Data 2): one set for Escherichia coli DHFR in its apo state and two inhibitor-bound states (Lu et al. 2026), and another set for the pre- and post-fusion stabilized states of the herpes simplex virus 1 (HSV-1) glycoprotein B (gB) (Roark et al. 2025). In both cases, we collected the data using a Bruker MaXis II LC-QTOF mass spectrometer. Both datasets were processed using PIGEON-derived peptide lists (Lu et al. 2026) in HDExaminer. State-specific HXMS files were then generated using PFLink: apo, methotrexate (MTX)-bound, and trimethoprim (TMP)-bound for DHFR; and pre- and post-fusion for HSV-1 gB. The DHFR files were generated with the fine match option enabled, and include the uncentroided fine structures from the raw mass spectra. The HSV-1 gB HXMS files contain bimodal spectra. Examples of PTMs can be found in the DynamX supporting files (Supplementary Data 1).

3. Discussion

In this article, we introduced HXMS and PFLink: a standardized, lightweight, and human-readable HX-MS data format, and a software package to convert HX-MS data to this format. In designing the HXMS format, we drew from the most successful elements of protein structure and genomics data formats to establish a flexible framework for HX-MS data inspection, storage, and sharing. The key advantage of the HXMS data format is its inclusion of isotopic mass envelopes and all other data necessary for quantitative and high-resolution data analysis. PFLink preserves all features of the data upon conversion to HXMS: experimental and biological replicates; PTMs; multimodal distributions; all measurements, including fully deuterated samples; and, when necessary or preferable, a mean deuteration-level data representation, and/or an uncentroided data representation.

While other HX-MS data formats are widely in use, these can often only be used with specific mass spectrometers and rely on mean deuteration representations, which fail to capture the full complexity of HX-MS data. The lack of standardization in the field, combined with the large size of raw HX-MS data files, limits the extraction of valuable information from past HX-MS studies. By contrast, the HXMS format is compatible with major commercial and academic HX-MS analysis software packages, including those enabling accurate and quantitative determination of high-resolution ensemble energies from HX-MS data (Kan et al. 2013, Gessner et al. 2017, Saltzberg et al. 2017, Babić, Kazazić and Smith 2019, Skinner et al. 2019, Salmas and Borysik 2021, Smit et al. 2021, Stofella et al. 2022, Lu et al. 2025, Puchała et al. 2025, Lu et al. 2026).

While the HXMS format aims to be a unified format for sharing HX-MS data, it is not intended to replace information-dense raw data files, but rather to be complementary to them. We strongly recommend that practitioners upload all raw data to repositories such as ProteomeXchange (Vizcaíno et al. 2014, Deutsch et al. 2023), while including HXMS files as supplementary data files with their publications. We hope that commercial and academic vendors of HX-MS analysis software packages will onboard HXMS format export capability.

Because upstream processing of raw HX-MS data directly impacts peptide selection and isotopic mass envelope extraction, vendor-specific handling can obscure how the data were processed. We address this by adding the MATCH section to the HXMS format, which ensures transparency regarding vendor-specific biases. The full traceability of peak assignments will allow for the review of isotopic mass envelope processing for debugging and other purposes without the need for vendor-specific software, greatly increasing the accessibility of the data. The MATCH section maintains a human-readable format to further assist with the inspection and debugging process.

Overall, the HXMS format will improve data parsing, analysis, sharing, and storage for the HX-MS community and enable future machine learning and integrated structural biology applications that require large amounts of HX-MS data from many practitioners. As MS technology develops, and the needs of our HX-MS community grow and change, the HXMS data format will evolve to accommodate HX-MS theoreticians, practitioners, and software/technology developers, as well as newcomers to the field.

Supplementary Material

btag113_Supplementary_Data
btag113_supplementary_data.zip (16.3MB, zip)

Acknowledgements

We acknowledge HX-MS data collection by Andrew Reckers for DHFR and Dr Malcolm Wells for HSV-1 gB, and software testing by Savannah McBride and other members of the Glasgow Lab, Dr Andrea Piserchio, Dr Rinat Abzalimov, and discussions with Vlad Sarpe (Trajan) and Dr Yoshitomo Hamuro (Johnson & Johnson).

Contributor Information

Kyle C Weber, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States.

Chenlin Lu, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States.

Roberto Vera Alvarez, Omics Informatics LLC, Honolulu, HI 96813, United States.

Bruce D Pascal, Omics Informatics LLC, Honolulu, HI 96813, United States.

Anum Glasgow, Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, United States.

Author contributions

Kyle Weber (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Software [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Chenlin Lu (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Investigation [equal], Methodology [equal], Software [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Roberto Vera Alvarez (Investigation [supporting], Software [supporting]), Bruce D. Pascal (Investigation [supporting], Software [supporting], Writing—review & editing [supporting]), and Anum Glasgow (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Funding acquisition [equal], Investigation [equal], Methodology [equal], Project administration [equal], Resources [equal], Software [equal], Supervision [equal], Validation [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal])

Supplementary material

Supplementary material is available at Bioinformatics online.

Conflict of interests

None declared.

Funding

This work has been supported by the National Institutes of Health (R35GM157185) and a National Science Foundation Graduate Research Fellowship to KCW.

Data availability

The data underlying this article are available in the online supplementary material section. This includes populated HXMS files and the input data used to produce them across various supported software formats. A blank custom format CSV is also included for practitioners to populate with key experimental data and results. The previously published ecDHFR data underlying this article are available in the ProteomeXchange Consortium (PRIDE) at https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD057539, and can be accessed with identifier PXD057539 (Lu and Glasgow 2024). The previously published HSV-1 gB data underlying this article are also available in Zenodo at https://doi.org/10.5281/zenodo.17354096, and can be accessed with DOI 10.5281/zenodo.17354096 (Wells and Glasgow 2025). The code and documentation for PFLink are available in Hugging Face at https://huggingface.co/spaces/glasgow-lab/PFLink/tree/main. The PFLink web application can be accessed at https://huggingface.co/spaces/glasgow-lab/PFLink.

References

  1. Babić D, Kazazić S, Smith DM.  Resolution of protein hydrogen/deuterium exchange by fitting amide exchange probabilities to the peptide isotopic envelopes. Rapid Commun Mass Spectrom  2019;33:1248–57. [DOI] [PubMed] [Google Scholar]
  2. Chalmers MJ, Busby SA, Pascal BD  et al.  Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Anal Chem  2006;78:1005–14. [DOI] [PubMed] [Google Scholar]
  3. Deutsch EW, Bandeira N, Perez-Riverol Y  et al.  The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res  2023; 51: D1539–D1548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Englander SW, Kallenbach NR.  Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q Rev Biophys  1983;16:521–655. [DOI] [PubMed] [Google Scholar]
  5. Englander SW, Mayne L, Kan Z-Y  et al.  Protein folding—how and why: by hydrogen exchange, fragment separation, and mass spectrometry. Annu Rev Biophys  2016;45:135–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gertsman I, Gan L, Guttman M  et al.  An unexpected twist in viral capsid maturation. Nature  2009;458:646–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gessner C, Steinchen W, Bédard S  et al.  Computational method allowing Hydrogen-Deuterium exchange mass spectrometry at single amide resolution. Sci Rep  2017;7:3789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Glasgow A, Hobbs HT, Perry ZR  et al.  Ligand-specific changes in conformational flexibility mediate long-range allostery in the Lac repressor. Nat Commun  2023;14:1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gramlich M, Hays HCW, Crichton S  et al.  HDX-MS for epitope characterization of a therapeutic ANTIBODY candidate on the calcium-binding protein Annexin-A1. Antibodies  2021;10:11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hamuro Y.  Determination of equine cytochrome c backbone amide hydrogen/deuterium exchange rates by mass spectrometry using a wider time window and isotope envelope. J Am Soc Mass Spectrom  2017;28:486–97. [DOI] [PubMed] [Google Scholar]
  11. Hamuro Y.  Interpretation of hydrogen/deuterium exchange mass spectrometry. J Am Soc Mass Spectrom  2024;35:819–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jia R, Bradshaw RT, Calvaresi V  et al.  Integrating hydrogen deuterium exchange–mass spectrometry with molecular simulations enables quantification of the conformational populations of the sugar transporter XylE. J Am Chem Soc  2023;145:7768–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kan Z, Ye X, Skinner JJ  et al.  ExMS2: an integrated solution for hydrogen–deuterium exchange mass spectrometry data analysis. Anal Chem  2019;91:7474–81. [DOI] [PubMed] [Google Scholar]
  14. Kan Z-Y, Walters BT, Mayne L  et al.  Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis. Proc Natl Acad Sci  2013;110:201315532. [Google Scholar]
  15. Kish M, Smith V, Lethbridge N  et al.  Online fully automated system for hydrogen/Deuterium-Exchange mass spectrometry with millisecond time resolution. Anal Chem  2023;95:5000–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lu C, Glasgow A. PIGEON-FEATHER datasets, extracting site-resolved energetic information from HX/MS experiments. ProteomeXchange Consortium (PRIDE). PXD057539, 2024. https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD057539
  17. Lu C, Weber KC, McBride SK  et al. A machine learning method for calculating highly localized protein stabilities. bioRxiv. 10.21.683809, 2025.
  18. Lu C, Wells ML, Reckers A  et al.  Site-resolved energetic information from HX–MS experiments. Nat Chem Biol  2026;22:307–17. [DOI] [PubMed] [Google Scholar]
  19. Masson GR, Burke JE, Ahn NG  et al.  Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat Methods  2019;16:595–602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pascal BD, Willis S, Lauer JL  et al.  HDX workbench: software for the analysis of H/D exchange MS data. J Am Soc Mass Spectrom  2012;23:1512–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Puchała W, Kistowski M, Zhukova L  et al.  HRaDeX: r package and web server for computing high-resolution deuterium uptake rates for HDX–MS data. J Proteome Res  2025;24:1688–700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roark RS, Schaub AJ, Shi W  et al.  Prefusion structure, evasion and neutralization of HSV-1 glycoprotein B. Nat Microbiol  2025;10:2966–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Salmas RE, Borysik AJ.  HDXmodeller: an online webserver for high-resolution HDX-MS with auto-validation. Commun Biol  2021;4:199–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Saltzberg DJ, Broughton HB, Pellarin R  et al.  A residue-resolved bayesian approach to quantitative interpretation of hydrogen–deuterium exchange from mass spectrometry: application to characterizing protein–ligand interactions. J Phys Chem B  2017;121:3493–501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shaw AL, Parson MAH, Truebestein L  et al.  ATP-competitive and allosteric inhibitors induce differential conformational changes at the autoinhibitory interface of Akt1. Structure  2023;31:343–54.e3. [DOI] [PubMed] [Google Scholar]
  26. Skinner SP, Radou G, Tuma R  et al.  Estimating constraints for protection factors from HDX-MS data. Biophys J  2019;116:1194–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smit JH, Krishnamurthy S, Srinivasu BY  et al.  Probing universal protein dynamics using hydrogen–deuterium exchange mass spectrometry-derived residue-level Gibbs free energy. Anal Chem  2021;93:12840–7. [DOI] [PubMed] [Google Scholar]
  28. Stofella M, Skinner SP, Sobott F  et al.  High-Resolution hydrogen–deuterium protection factors from sparse mass spectrometry data validated by nuclear magnetic resonance measurements. J Am Soc Mass Spectrom  2022;33:813–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vizcaíno JA, Deutsch EW, Wang R  et al.  ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat Biotechnol  2014;32:223–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wales TE, Fadgen KE, Gerhardt GC  et al.  High-speed and high-resolution UPLC separation at zero degrees Celsius. Anal Chem  2008;80:6815–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wales TE, Pajak A, Roeselová A  et al.  Resolving chaperone-assisted protein folding on the ribosome at the peptide level. Nat Struct Mol Biol  2024;31:1888–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang LC, Morgan LK, Godakumbura P  et al.  The inner membrane histidine kinase EnvZ senses osmolality via helix‐coil transitions in the cytoplasm. Embo J  2012;31:2648–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wei H, Mo J, Tao L  et al.  Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discov Today  2014;19:95–102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wells M, Glasgow A.  HDX/MS dataset for prefusion structure, evasion and neutralization of HSV-1 glycoprotein B. Zenodo, 2025. 10.5281/zenodo.17354096 [DOI]
  35. Wells ML, Lu C, Sultanov D  et al. Distinct energetic blueprints diversify function of conserved protein folds. bioRxiv. 04.02.646877, 2025.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

  1. Wells M, Glasgow A.  HDX/MS dataset for prefusion structure, evasion and neutralization of HSV-1 glycoprotein B. Zenodo, 2025. 10.5281/zenodo.17354096 [DOI]

Supplementary Materials

btag113_Supplementary_Data
btag113_supplementary_data.zip (16.3MB, zip)

Data Availability Statement

The data underlying this article are available in the online supplementary material section. This includes populated HXMS files and the input data used to produce them across various supported software formats. A blank custom format CSV is also included for practitioners to populate with key experimental data and results. The previously published ecDHFR data underlying this article are available in the ProteomeXchange Consortium (PRIDE) at https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD057539, and can be accessed with identifier PXD057539 (Lu and Glasgow 2024). The previously published HSV-1 gB data underlying this article are also available in Zenodo at https://doi.org/10.5281/zenodo.17354096, and can be accessed with DOI 10.5281/zenodo.17354096 (Wells and Glasgow 2025). The code and documentation for PFLink are available in Hugging Face at https://huggingface.co/spaces/glasgow-lab/PFLink/tree/main. The PFLink web application can be accessed at https://huggingface.co/spaces/glasgow-lab/PFLink.

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